Non-caloric sweeteners and methods for synthesizing

ABSTRACT

Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebausoside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/697,628, filed Sep. 7, 2017, entitled “NON-CALORIC SWEETENERS ANDMETHODS FOR SYNTHESIZING,” which is a continuation of U.S. patentapplication Ser. No. 14/873,481, filed Oct. 2, 2015, entitled“NON-CALORIC SWEETENERS AND METHODS FOR SYNTHESIZING,” which claimspriority to U.S. Provisional Patent Application No. 62/059,498, filedOct. 3, 2014, entitled “NON-CALORIC SWEETENERS AND METHODS FORSYNTHESIZING,” and to U.S. Provisional Patent Application No.62/098,929, filed Dec. 31, 2014, entitled “NONCALORIC SWEETENERS ANDMETHODS FOR SYNTHESIZING,” the disclosures of each of which are herebyincorporated by reference in their entirety.

STATEMENT IN SUPPORT FOR FILING A SEQUENCE LISTING

A computer readable form of the Sequence Listing containing the filenamed “C149770007US22-SEQ-ZJG.txt”, which is 60,824 bytes in size (asmeasured in MICROSOFT WINDOWS® EXPLORER), is provided herewith and isherein incorporated by reference. This Sequence Listing consists of SEQID NOs:1-12.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to natural sweeteners. Moreparticularly, the present disclosure relates to a non-caloric sweetenerand methods for synthesizing the non-caloric sweetener.

Steviol glycosides are natural products isolated from Stevia rebaudianaleaves. Steviol glycosides are widely used as high intensity,low-calorie sweeteners and are significantly sweeter than sucrose. Asnatural sweeteners, different steviol glucosides have different degreesof sweetness and after-taste. The sweetness of steviol glycosides issignificantly higher than that of sucrose. For example, stevioside is100-150 times sweeter than sucrose with bitter after-taste. RebaudiosideC is between 40-60 times sweeter than sucrose. Dulcoside A is about 30times sweeter than sucrose.

Naturally occurring steviol glycosides share the same basic steviolstructure, but differ in the content of carbohydrate residues (e.g.,glucose, rhamnose and xylose residues) at the C13 and C19 positions.Steviol glycosides with known structures include, steviol, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside E, rebaudioside F and dulcoside A (see e.g., Table 1).Other steviol glycosides are rebaudioside M, rebaudioside N andrebaudioside O.

TABLE 1 Steviol glycosides. Molecular Molecular Name Structure FormulaWeight Steviol

C₂₀H₃₀O₃ 318 Stevioside

C₃₈H₆₀O₁₈ 804 Rebaudioside A

C₄₄H₇₀O₂₃ 966 Rebaudioside- B

C₃₈H₆₀O₁₈ 804 Rebaudioside C

C₄₄H₇₀O₂₂ 950 Rebaudioside D

C₅₀H₈₀O₂₈ 1128 Rebaudioside E

C₄₄H₇₀O₂₃ 966 Rebaudioside F

C₄₃H₆₈O₂₂ 936 Rebaudioside G

C₃₈H₆₀O₁₈ 804 Rebaudioside D3

C₅₀H₈₀O₂₈ 1128 Rebaudioside KA

C₃₈H₆₀O₁₈ 804 Dulcoside A

C₃₈H₆₀O₁₇ 788

On a dry weight basis, stevioside, rebaudioside A, rebaudioside C, anddulcoside A, account for 9.1, 3.8, 0.6, and 0.3% of the total weight ofthe steviol glycosides in the leaves, respectively, while the othersteviol glycosides are present in much lower amounts. Extracts from theStevia rebaudiana plant are commercially available, which typicallycontain stevioside and rebaudioside A as primary compounds. The othersteviol glycosides typically are present in the stevia extract as minorcomponents. For example, the amount of rebaudioside A in commercialpreparations can vary from about 20% to more than 90% of the totalsteviol glycoside content, while the amount of rebaudioside B can beabout 1-2%, the amount of rebaudioside C can be about 7-15%, and theamount of rebaudioside D can be about 2% of the total steviolglycosides.

The majority of steviol glycosides are formed by several glycosylationreactions of steviol, which are typically catalyzed by theUDP-glycosyltransferases (UGTs) using uridine 5′-diphosphoglucose(UDP-glucose) as a donor of the sugar moiety. UGTs in plants make up avery diverse group of enzymes that transfer a glucose residue fromUDP-glucose to steviol. For example, glycosylation of the C-3′ of theC-13-O-glucose of stevioside yields rebaudioside A; and glycosylation ofthe C-2′ of the 19-O-glucose of the stevioside yields rebaudioside E.Further glycosylation of rebaudioside A (at C-2′-19-O-glucose) orrebaudioside E (at C-3′-13-O-glucose) produces rebaudioside D. (FIG. 1Aand FIG. 1B).

Alternative sweeteners are receiving increasing attention due toawareness of many diseases in conjunction with the consumption ofhigh-sugar foods and beverages. Although artificial sweeteners areavailable, many artificial sweeteners such as dulcin, sodium cyclamateand saccharin have been banned or restricted by some countries due toconcerns over their safety. Therefore, non-caloric sweeteners of naturalorigin are becoming increasingly popular. One of the main obstacles forthe widespread use of stevia sweeteners are their undesirable tasteattributes. Accordingly, there exists a need to develop alternativesweeteners and methods for their production to provide the bestcombination of sweetness potency and flavor profile.

SUMMARY OF THE DISCLOSURE

The present disclosure relates generally to natural sweeteners. Moreparticularly, the present disclosure relates to non-caloric sweetenersand methods for synthesizing the non-caloric sweeteners.

Synthetic Rebaudioside V.

In one aspect, the present disclosure is directed to a syntheticrebaudioside (rebaudioside V) consisting of a chemical structure:

Synthetic Rebaudioside W.

In one aspect, the present disclosure is directed to a syntheticrebaudioside (rebaudioside W) consisting of a chemical structure:

Method of Producing Rebaudioside V from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rebaudioside G. The method includespreparing a reaction mixture comprising rebaudioside G, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), and a HV1UDP-glycosyltransferase, with or without sucrose synthase (SUS); andincubating the reaction mixture for a sufficient time to producerebaudioside V, wherein a glucose is covalently coupled to therebaudioside G to produce rebaudioside V.

Method of Producing Rebaudioside V from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rebaudioside G. The method includespreparing a reaction mixture comprising rebaudioside G, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), a uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a uridine diphospho glycosyltransferase (EUGT11), aUDP-glycosyltransferase-Sucrose synthase (SUS) fusion enzyme, with orwithout sucrose synthase (SUS); and incubating the reaction mixture fora sufficient time to produce rebaudioside V, wherein a glucose iscovalently coupled to the rebaudioside G to produce rebaudioside V.

Method of Producing Rebaudioside V from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rebaudioside KA. The method includespreparing a reaction mixture comprising rebaudioside KA, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), a uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1; SEQ ID NO:1) and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase (SUS); and incubating the reaction mixture for asufficient time to produce rebaudioside V, wherein a glucose iscovalently coupled to the rebaudioside KA to produce rebaudioside V.

Method of Producing Rebaudioside V from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), uridine diposphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1), HV1 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase (SUS); and incubating the reaction mixture for asufficient time to produce rebaudioside V, wherein a glucose iscovalently coupled to the rubusoside to produce rebaudioside KA.Continually, a glucose is covalently coupled to the rebaudioside KA toproduce rebaudioside V. A glucose is covalently coupled to therubusoside to produce rebaudioside G. Continually, a glucose iscovalently coupled to the rebaudioside G to produce rebaudioside V.

Method of Producing Rebaudioside V from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), uridine diposphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1), EUGT11 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside V, wherein a glucose is covalently coupledto the rubusoside to produce rebaudioside KA and a glucose is covalentlycoupled to the rebaudioside KA to produce rebaudioside V. A glucose iscovalently coupled to the rubusoside to product rebaudioside G and aglucose is covalently coupled to the rebaudioside G to producerebaudioside V.

Method of producing Rebaudioside W from Rebaudioside V.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside V. The method includespreparing a reaction mixture comprising rebaudioside V, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), a uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1) and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside W, wherein a glucose is covalently coupledto the rebaudioside V to produce rebaudioside W.

Method of Producing Rebaudioside W from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside G. The method includespreparing a reaction mixture comprising rebaudioside G, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), uridine diposphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1), aUDP-glycosyltransferase-Sucrose synthase fusion enzyme and a HV1; withor without sucrose synthase; and incubating the reaction mixture for asufficient time to produce rebaudioside W, wherein a glucose iscovalently coupled to the rebaudioside G to produce rebaudioside V byHV1. Continually, a glucose is covalently coupled to the rebaudioside Vto produce rebaudioside W by UGT76G1.

Method of Producing Rebaudioside W from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside G. The method includespreparing a reaction mixture comprising rebaudioside G, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), uridine diphosphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of a UGT76G1, an EUGT11, and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; and incubatingthe reaction mixture for a sufficient time to produce rebaudioside W,wherein a glucose is covalently coupled to the rebaudioside G to producerebaudioside V by EUGT11. Continually, a glucose is covalently coupledto the rebaudioside V to produce rebaudioside W by UGT76G1.

Method of Producing Rebaudioside W from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside KA. The method includespreparing a reaction mixture comprising rebaudioside KA; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); a uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1), and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside W, wherein a glucose is covalently coupledto the rebaudioside KA to produce rebaudioside V. Continually, a glucoseis covalently coupled to the rebaudioside V to produce rebaudioside W.

Method of Producing of Rebaudioside W from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), uridine diphosphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of an UGT76G1, a HV1, and a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, with or without sucrose synthase; and incubatingthe reaction mixture for a sufficient time to produce rebaudioside W.

Method of Producing of Rebaudioside W from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), uridine diphosphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of an UGT76G1, an EUGT11, and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside W.

Method of Producing a Mixture of Stevioside and Rebaudioside KA fromRubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a mixture of stevioside and rebaudioside KA fromrubusoside. The method includes preparing a reaction mixture comprisingrubusoside, substrates selected from the group consisting of sucrose,uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose),a UDP-glycosyltransferase selected from the group consisting of EUGT11and a UDP-glycosyltransferase-Sucrose synthase fusion enzyme, with orwithout sucrose synthase; and incubating the reaction mixture for asufficient time to produce a mixture of stevioside and rebaudioside KA,wherein a glucose is covalently coupled to C2′-19-O-glucose ofrubusoside to produce rebaudioside KA; a glucose is covalently coupledto C2′-13-O-glucose of rubusoside to produce stevioside.

Method of Producing Rebaudioside KA from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a rebaudioside KA from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), and HV1, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside KA, wherein a glucose is covalently coupledto the C2′-19-O-glucose of rubusoside to produce a rebaudioside KA.

Method of Producing Rebaudioside G from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a rebaudioside G from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferaseselected from the group consisting of UGT76G1 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside G, wherein a glucose is covalently coupledto the C3′-13-O-glucose of rubusoside to produce a rebaudioside G.

Method of Producing Rebaudioside E from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rebaudioside KA. The method includespreparing a reaction mixture comprising rebaudioside KA, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose) and HV1UDP-glycosyltransferase, with or without sucrose synthase; andincubating the reaction mixture for a sufficient time to producerebaudioside E, wherein a glucose is covalently coupled to the C2′13-O-glucose of rebaudioside KA to produce rebaudioside E.

Method of Producing Rebaudioside E from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rebaudioside KA. The method includespreparing a reaction mixture comprising rebaudioside KA, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferaseselected from the group consisting of an EUGT11 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside E, wherein a glucose is covalently coupledto the C2′ 13-O-glucose of rebaudioside KA to produce rebaudioside E.

Method of Producing Rebaudioside E from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), and a UDP-glycosyltransferasefrom the group of EUGT11 and a UDP-glycosyltransferase-Sucrose synthesisfusion enzyme, with or without sucrose synthase; and incubating thereaction mixture for a sufficient time to produce rebaudioside E,wherein a glucose is covalently coupled to rubusoside to produce amixture of rebaudioside KA and stevioside. Continually, a glucose iscovalently coupled to rebaudioside KA and stevioside to producerebaudioside E.

Method of Producing Rebaudioside E from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose) and HV1UDP-glycosyltransferase, with or without sucrose synthase; incubatingthe reaction mixture for a sufficient time to produce rebaudioside E,wherein a glucose is covalently coupled to the rubusoside to producerebaudioside KA; and further incubating the rebaudioside KA with HV1 toproduce rebaudioside E.

Method of Producing Rebaudioside D3 from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D3 from rubusoside. The method includespreparing a reaction mixture comprising rubusoside, substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferasefrom the group of an EUGT11 and a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, with or without sucrose synthase; incubating thereaction mixture for a sufficient time to produce a mixture ofstevioside and rebaudioside D3, wherein a glucose is covalently coupledto the rubusoside to produce a mixture of stevioside and rebaudiosideKA; further incubating the mixture of stevioside and rebaudioside KAwith EUGT11 to produce rebaudioside E, wherein a glucose is covalentlycoupled to the stevioside and the rebaudioside KA to producerebaudioside E; and further incubating the rebaudioside E with EUGT11 toproduce rebaudioside D3, wherein a glucose is covalently coupled to therebaudioside E to produce rebaudioside D3.

Method of Producing Rebaudioside D3 from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D3 from rebaudioside KA. The method includespreparing a reaction mixture comprising rebaudioside KA, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferaseselected from the group consisting of an EUGT11 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; incubating the reaction mixture for a sufficient timeto produce rebaudioside D3, wherein a glucose is covalently coupled tothe rebaudioside KA to produce rebaudioside E; further incubating themixture of rebaudioside E with EUGT11 to produce rebaudioside D3,wherein a glucose is covalently coupled to the rebaudioside E to producerebaudioside D3.

Method of Producing Rebaudioside Z from Rebaudioside E.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside Z from rebaudioside E. The method includespreparing a reaction mixture comprising rebaudioside E, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), HV1 and sucrose synthase;incubating the reaction mixture for a sufficient time to producerebaudioside Z, wherein a glucose is covalently coupled to theC2′-13-O-glucose of rebaudioside E to produce rebaudioside Z1. A glucoseis covalently coupled to C2′-19-O-glucose of rebaudioside E to producerebaudioside Z2.

Method of Producing Rebaudioside M from Rebaudioside D.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside M from rebaudioside D. The method includespreparing a reaction mixture comprising rebaudioside D, substratesselected from the group consisting of sucrose, uridine diphosphate(UDP), uridine diphosphate-glucose (UDP-glucose), and combinationsthereof, and a UDP-glycosyltransferase selected from the groupconsisting of UGT76G1, a UDP-glycosyltransferase-Sucrose synthase fusionenzyme, and combinations thereof, with or without sucrose synthase; andincubating the reaction mixture for a sufficient time to producerebaudioside M, wherein a glucose is covalently coupled to therebaudioside D to produce rebaudioside M.

Method of Producing Rebaudioside D and Rebaudioside M from Stevioside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D and rebaudioside M from stevioside. Themethod includes preparing a reaction mixture comprising stevioside,substrates selected from the group consisting of sucrose, uridinediphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), andcombinations thereof, and a UDP-glycosyltransferase selected from thegroup consisting of HV1, UGT76G1, a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, and combinations thereof, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside M. In certain embodiments, a glucose iscovalently coupled to the stevioside to produce rebaudioside A and/orrebaudioside E. Continually, a glucose is covalently coupled to therebaudioside A and/or rebaudioside E to produce rebaudioside D, and aglucose is covalently coupled to the rebaudioside D to producerebaudioside M.

Method of Producing Rebaudioside D and Rebaudioside M from RebaudiosideA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D and rebaudioside M from rebaudioside A. Themethod includes preparing a reaction mixture comprising rebaudioside A,substrates selected from the group consisting of sucrose, uridinediphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), andcombinations thereof, and a UDP-glycosyltransferase selected from thegroup consisting of HV1, UGT76G1, a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, and combinations thereof, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside M, wherein a glucose is covalently coupledto the rebaudioside A to produce rebaudioside D, and a glucose iscovalently coupled to the rebaudioside D to produce rebaudioside M.

Method of Producing Rebaudioside D and Rebaudioside M from RebaudiosideE.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D and rebaudioside M from rebaudioside E. Themethod includes preparing a reaction mixture comprising rebaudioside E,substrates selected from the group consisting of sucrose, uridinediphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), andcombinations thereof, and a UDP-glycosyltransferase selected from thegroup consisting of an UGT76G1, a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, and combinations thereof, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside M, wherein a glucose is covalently coupledto the rebaudioside E to produce rebaudioside D, and wherein a glucoseis covalently coupled to the rebaudioside D to produce rebaudioside M.

In another aspect, the present disclosure is directed to an orallyconsumable product comprising a sweetening amount of a rebaudiosideselected from rebaudioside V, rebaudioside W, rebaudioside G,rebaudioside KA, rebaudioside M, and combinations thereof, wherein theorally consumable product is selected from the group consisting of abeverage product and a consumable product.

In another aspect, the present disclosure is directed to a beverageproduct comprising a sweetening amount of a rebaudioside selected fromrebaudioside V, rebaudioside W, rebaudioside G, rebaudioside KA,rebaudioside M, and combinations thereof. The rebaudioside is present inthe beverage product at a concentration of about 5 ppm to about 100 ppm.In some embodiments, low concentrations of rebaudioside, e.g., below 100ppm, has an equivalent sweetness to sucrose solutions havingconcentrations between 10,000 and 30,000 ppm.

In another aspect, the present disclosure is directed to a consumableproduct comprising a sweetening amount of a rebaudioside selected fromrebaudioside V, rebaudioside W, rebaudioside G, rebaudioside KA,rebaudioside M, and combinations thereof. The rebaudioside is present inthe consumable product at a concentration of about 5 ppm to about 100ppm. In some embodiments, low concentrations of rebaudioside, e.g.,below 100 ppm, has an equivalent sweetness to sucrose solutions havingconcentrations between 10,000 and 30,000 ppm.

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In certain embodiments that can be combined with any of the precedingembodiments, the rebaudioside V or the rebaudioside W or therebaudioside G or the rebaudioside KA or the rebaudioside M can be theonly sweetener, and the product has a sweetness intensity equivalent toabout 1% to about 4% (w/v-%) sucrose solution. In certain embodimentsthat can be combined with any of the preceding embodiments, the orallyconsumable product further can include an additional sweetener, wherethe product has a sweetness intensity equivalent to about 1% to about10% (w/v-%) sucrose solution. In certain embodiments that can becombined with any of the preceding embodiments, every sweeteningingredient in the product can be a high intensity sweetener. In certainembodiments that can be combined with any of the preceding embodiments,every sweetening ingredient in the product can be a natural highintensity sweetener. In certain embodiments that can be combined withany of the preceding embodiments, the additional sweetener can be one ormore sweeteners selected from a stevia extract, a steviol glycoside,stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudiosideD, rebaudioside D3, rebaudioside E, rebaudioside F, rebaudioside G,rebaudioside KA, rebaudioside M, dulcoside A, rubusoside,steviolbioside, sucrose, high fructose corn syrup, fructose, glucose,xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol,inositol, AceK, aspartame, neotame, sucralose, saccharine, naringindihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC),rubusoside, mogroside IV, siamenoside I, mogroside V, monatin,thaumatin, monellin, brazzein, L-alanine, glycine, Lo Han Guo,hernandulcin, phyllodulcin, trilobtain, and combinations thereof. Incertain embodiments that can be combined with any of the precedingembodiments, the beverage product and consumable product can furtherinclude one or more additives selected from a carbohydrate, a polyol, anamino acid or salt thereof, a poly-amino acid or salt thereof, a sugaracid or salt thereof, a nucleotide, an organic acid, an inorganic acid,an organic salt, an organic acid salt, an organic base salt, aninorganic salt, a bitter compound, a flavorant, a flavoring ingredient,an astringent compound, a protein, a protein hydrolysate, a surfactant,an emulsifier, a flavonoids, an alcohol, a polymer, and combinationsthereof. In certain embodiments that can be combined with any of thepreceding embodiments, the rebaudioside V has a purity of about 50% toabout 100% by weight before it is added into the product. In certainembodiments that can be combined with any of the preceding embodiments,the W has a purity of about 50% to about 100% by weight before it isadded into the product. In certain embodiments that can be combined withany of the preceding embodiments, the rebaudioside V in the product is arebaudioside V polymorph or amorphous rebaudioside V. In certainembodiments that can be combined with any of the preceding embodiments,the rebaudioside V in the product is a rebaudioside V stereoisomer. Incertain embodiments that can be combined with any of the precedingembodiments, the rebaudioside W in the product is a rebaudioside Wpolymorph or amorphous rebaudioside W. In certain embodiments that canbe combined with any of the preceding embodiments, the rebaudioside W inthe product is a rebaudioside W stereoisomer.

Other aspects of the present disclosure relate to a method of preparinga beverage product and a consumable product by including synthesizedrebaudioside selected from rebaudioside V, rebaudioside W, rebaudiosideKA, rebaudioside M, and rebaudioside G into the product or into theingredients for making the beverage product and the consumable product,where rebaudioside selected from rebaudioside V, rebaudioside W,rebaudioside KA, rebaudioside M, and rebaudioside G is present in theproduct at a concentration of from about 5 ppm to about 100 ppm. Otheraspects of the present disclosure relate to a method for enhancing thesweetness of a beverage product and a consumable product by adding fromabout 5 ppm to about 100 ppm of synthesized rebaudioside selected fromrebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, andrebaudioside G into the beverage product and the consumable product,where the added synthesized rebaudioside selected from rebaudioside V,rebaudioside W, rebaudioside KA, rebaudioside M, and rebaudioside Genhances the sweetness of the beverage product and the consumableproduct, as compared to a corresponding a beverage product and aconsumable product lacking the synthesized rebaudioside selected fromrebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, andrebaudioside G.

In certain embodiments that can be combined with any of the precedingembodiments, rebaudioside V is the only sweetener, and the product has asweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrosesolution. In certain embodiment that can be combined with any of theproceeding embodiments, rebaudioside KA is the only sweetener, and theproduct has a sweetness intensity equivalent to about 1% to about 4%(w/v-%) sucrose solution. In certain embodiments that can be combinedwith any of the proceeding embodiments, rebaudioside G is the onlysweetener, and the product has a sweetness intensity equivalent to about1% to about 4% (w/v-%) sucrose solution. In certain embodiments that canbe combined with any of the preceding embodiments, rebaudioside W is theonly sweetener, and the product has a sweetness intensity equivalent toabout 1% to about 4% (w/v-%) sucrose solution. In certain embodimentsthat can be combined with any of the preceding embodiments, rebaudiosideM is the only sweetener, and the product has a sweetness intensityequivalent to about 1% to about 4% (w/v-%) sucrose solution. In certainembodiments that can be combined with any of the preceding embodiments,the method further includes adding an additional sweetener, where theproduct has a sweetness intensity equivalent to about 1% to about 10%(w/v-%) sucrose solution.

Other aspects of the present disclosure relate to a method for preparinga sweetened beverage product or a sweetened consumable product by: a)providing a beverage product or a consumable product containing one ormore sweetener; and b) adding from about 5 ppm to about 100 ppm of asynthesized rebaudioside selected from rebaudioside V, rebaudioside W,rebaudioside KA, rebaudioside M, and rebaudioside G, and combinationsthereof into the beverage product or the consumable product.

In certain embodiments that can be combined with any of the precedingembodiments, the method further includes adding one or more additives tothe beverage product or the consumable product. In certain embodimentsthat can be combined with any of the preceding embodiments, the orallyconsumable product further contains one or more additives. In certainembodiments that can be combined with any of the preceding embodiments,the one or more additives are selected from a carbohydrate, a polyol, anamino acid or salt thereof, a poly-amino acid or salt thereof, a sugaracid or salt thereof, a nucleotide, an organic acid, an inorganic acid,an organic salt, an organic acid salt, an organic base salt, aninorganic salt, a bitter compound, a flavorant, a flavoring ingredient,an astringent compound, a protein, a protein hydrolysate, a surfactant,an emulsifier, a flavonoids, an alcohol, a polymer, and combinationsthereof. In certain embodiments that may be combined with any of thepreceding embodiments, every sweetening ingredient in the product is ahigh intensity sweetener. In certain embodiments that can be combinedwith any of the preceding embodiments, every sweetening ingredient inthe product is a natural high intensity sweetener. In certainembodiments that can be combined with any of the preceding embodiments,the sweetener is selected from a stevia extract, a steviol glycoside,stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudiosideD, rebaudioside D3, rebaudioside E, rebaudioside F, rebaudioside G,rebaudioside KA, rebaudioside M, dulcoside A, rubusoside,steviolbioside, sucrose, high fructose corn syrup, fructose, glucose,xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol,inositol, AceK, aspartame, neotame, sucralose, saccharine, naringindihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC),rubusoside, mogroside IV, siamenoside I, mogroside V, monatin,thaumatin, monellin, brazzein, L-alanine, glycine, Lo Han Guo,hernandulcin, phyllodulcin, trilobtain, and combinations thereof. Incertain embodiments that can be combined with any of the precedingembodiments, the rebaudioside V has a purity of about 50% to about 100%by weight before it is added into the product. In certain embodimentsthat can be combined with any of the preceding embodiments, therebaudioside V in the product is a rebaudioside V polymorph or amorphousrebaudioside V. In certain embodiments that can be combined with any ofthe preceding embodiments, the rebaudioside W has a purity of about 50%to about 100% by weight before it is added into the product. In certainembodiments that can be combined with any of the preceding embodiments,the rebaudioside W in the product is a rebaudioside W polymorph oramorphous rebaudioside W.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIG. 1A and FIG. 1B depict a steviol glycosides biosynthesis pathwayfrom steviol.

FIG. 2 depicts SDS-PAGE analysis purified recombinant proteins indicatedby arrows: A: HV1, B: UGT76G1, C: EUGT11, D: AtSUS1, E: UGT76G1-SUS1(GS), F: EUGT11-SUS1 (EUS).

FIG. 3 depicts the HV1 catalysis reaction to produce rebaudioside KA(“Reb KA”) and rebaudioside E (“Reb E”) from rubusoside. A-C: showingthe HPLC retention times of rubusoside (“Rub”), stevioside (“Ste”) andrebaudioside E (“Reb E”) standards. Reb KA enzymatically produced by HV1alone at 6 hr (D), 12 hr (F) and 24 hours (H); Reb KA and Reb Eenzymatically produced by the UGT-SUS (HV1-AtSUS1) coupling system at 6hr (E), 12 hr (G) and 24 hr (I).

FIG. 4 depicts the conversion of Reb E to rebaudioside Z by HV1. (A):shows the HPLC retention time of rebaudioside E (“Reb E”). RebaudiosideZ (“Reb Z”) enzymatically produced by HV1 in the HV1-AtSUS1 couplingsystem at 3 hr (B), 7 hr (C), 24 hr (D) and 44 hr (E).

FIG. 5 depicts the conversion of Reb KA to Reb E by HV1. (A-B): show theHPLC retention times of rebaudioside KA (“Reb KA”) and rebaudioside E(“Reb E”) standards. Reb E enzymatically produced by HV1 alone at 12 hr(C); Reb E enzymatically produced by the UGT-SUS (HV1-AtSUS1) couplingsystem at 12 hr (D).

FIG. 6 depicts the EUGT11 catalysis reaction to produce Reb KA andstevioside from rubusoside. (A-F): show the HPLC retention times ofrubusoside (“Rub”), stevioside (“Ste”), rebaudioside G (“Reb G”),rebaudioside E (“Reb E”), rebaudioside D (“Reb D”) and rebaudioside D3(“Reb D3”) standards. Enzymatic reaction by EUGT11 alone at 12 hr (G)and 48 hr (J); enzymatic reaction by the UGT-SUS (EUGT11-AtSUS1)coupling system at 12 hr (H) and 48 hr (K); enzymatic reaction by EUSfusion protein at 12 hr (I) and 48 hr (L).

FIG. 7 depicts the conversion of Reb KA to Reb E and Reb D3 by EUGT11and EUS fusion proteins. (A-C): showing the HPLC retention times ofrebaudioside KA (“Reb KA”), rebaudioside E (“Reb E”), and rebaudiosideD3 (“Reb D3”) standards. Enzymatic reaction by EUGT11 alone at 12 hr (D)and 48 hr (G); enzymatic reaction by the UGT-SUS (EUGT11-AtSUS1)coupling system at 12 hr (E) and 48 hr (H); enzymatic reaction by EUSfusion protein at 12 hr (F) and 48 hr (I).

FIG. 8 depicts the UGT76G1 production of rebaudioside G in vitro. (A-B):show the HPLC retention times of rubusoside (“Rub”) and rebaudioside G(“Reb G”) standards. Enzymatic reaction by UGT76G1 alone at 12 hr (C)and 24 hr (F); enzymatic reaction by the UGT-SUS (EUGT11-AtSUS1)coupling system at 12 hr (D) and 24 hr (G); enzymatic reaction by GSfusion protein at 12 hr (E) and 48 hr (H).

FIG. 9 depicts the UGT76G1 catalysis reaction to produce the steviolglycosides Reb V and Reb W from rebaudioside KA. (A-D): show the HPLCretention times of rubusoside (“Rub”), rebaudioside D (“Reb D”),rebaudioside E (“Reb E”) and rebaudioside KA (“Reb KA”) standards.Enzymatic reaction by UGT76G1 alone at 6 hr (E) and 12 hr (H); enzymaticreaction by the UGT-SUS (UGT76G1-AtSUS1) coupling system at 6 hr (F) and12 hr (I); enzymatic reaction by GS fusion protein at 6 hr (G) and 12 hr(J).

FIG. 10 depicts the UGT76G1 conversion of Reb V to Reb W in vitro.(A-B): showing the HPLC retention times of Reb V and Reb W. (C):Enzymatic reaction by the UGT76G1-AtSUS1 coupling system at 6 hr.

FIG. 11 depicts the HV1 conversion of Reb G to Reb V. (A-C): showing theHPLC retention times of rebaudioside G (“Reb G”), rebaudioside A (“RebA”) and rebaudioside E (“Reb E”) standards. Enzymatic reaction by HV1alone at 12 hr (D) and 24 hr (F); enzymatic reaction by the UGT-SUS(HV1-AtSUS1) coupling system at 12 hr (E) and 24 hr (G).

FIG. 12 depicts the EUGT11 conversion of Reb G to Reb V. (A-D): showingthe HPLC retention times of rebaudioside G (“Reb G”), rebaudioside A(“Reb A”), rebaudioside E (“Reb E”) and rebaudioside D (“Reb D”)standards. Enzymatic reaction by EUGT11 alone at 12 hr (E) and 24 hr(H); enzymatic reaction by the UGT-SUS (EUGT11-AtSUS1) coupling systemat 12 hr (F) and 24 hr (I); enzymatic reaction by EUS fusion enzyme at12 hr (G) and 24 hr (J).

FIG. 13 depicts the in vitro production of Reb W from rubusosidecatalyzed by a combination of a recombinant HV1 polypeptide, arecombinant UGT76G1, a GS fusion enzyme, and a recombinant AtSUS1.(A-F): show the standards of rubusoside (“Rub”), stevioside (“Ste”),Rebaudioside G (“Reb G”), rebaudioside A (“Reb A”), Rebaudioside D (“RebD”) and rebaudioside E (“Reb E”). Reb W enzymatically produced by HV1,UGT76G1 and AtSUS1 at 6 hours (G), 12 hr (I) and 24 hr (K); Reb Wenzymatically produced by HV1 and GS fusion protein at 6 hours (H), 12hr (J) and 24 hr (L).

FIG. 14 depicts the in vitro production of Reb W from rubusosidecatalyzed by a combination of a recombinant EUGT11 polypeptide, arecombinant UGT76G1, a GS fusion enzyme, and a recombinant AtSUS1.(A-E): show the standards of rubusoside (“Rub”), stevioside (“Ste”),rebaudioside G (“Reb G”), rebaudioside E (“Reb E”) and rebaudioside D(“Reb D”). Reb W enzymatically produced by EUGT11, UGT76G1 and AtSUS1 at12 hours (F) and 48 hr (H); Reb W enzymatically produced by EUGT11 andGS fusion protein at 12 hours (G) and 48 hr (I).

FIG. 15 depicts the in vitro production of Reb W from Reb G catalyzed bya combination of a recombinant HV1 polypeptide, a recombinant UGT76G1, aGS fusion enzyme and a recombinant AtSUS1. A-D shows the standards ofrebaudioside G (“Reb G”), rebaudioside A (“Reb A”), Rebaudioside D (“RebD”), rebaudioside and rebaudioside E (“Reb E”). Reb V and Reb Wenzymatically produced by HV1, UGT76G1 and AtSUS1 at 6 hours (E), 12 hr(G) and 36 hr (I); Reb V and Reb W enzymatically produced by HV1 and GSfusion protein at 6 hours (F), 12 hr (H) and 36 hr (J).

FIG. 16 depicts the in vitro production of Reb W from Reb G catalyzed bya combination of a recombinant EUGT11 polypeptide, a recombinantUGT76G1, a GS fusion enzyme, and a recombinant AtSUS1. (A-D): show thestandards of rebaudioside G (“Reb G”), rebaudioside A (“Reb A”),rebaudioside E (“Reb E”) and rebaudioside D (“Reb D”). Reb Wenzymatically produced by EUGT11, UGT76G1 and AtSUS1 at 12 hours (E) and48 hr (G); Reb W enzymatically produced by EUGT11 and GS fusion proteinat 12 hours (F) and 48 hr (H).

FIG. 17 depicts the structures of Reb V and Reb G.

FIG. 18 depicts the key TOCSY and HMBC correlations of Reb V.

FIG. 19 depicts the structures of Reb W and Reb V.

FIG. 20 depicts the key TOCSY and HMBC correlations of Reb W.

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D depict the biosynthesispathway of steviol glycosides.

FIG. 22 depicts the in vitro production of Reb M from Reb D catalyzed byUGT76G1 and GS fusion enzyme. (A-B): showing the HPLC retention times ofrebaudioside D (“Reb D”) and rebaudioside M (“Reb M) standards.Enzymatic reaction by UGT76G1 alone at 3 hr (C) and 6 hr (F); enzymaticreaction by the UGT-SUS (UGT76G1-AtSUS1) coupling system at 3 hr (D) and6 hr (G); enzymatic reaction by the GS fusion enzyme at 3 hr (E) and 6hr (H).

FIG. 23 depicts the in vitro production of Reb D and Reb M from Reb Ecatalyzed by UGT76G1 and GS fusion enzyme. (A-C): showing the HPLCretention times of rebaudioside E (“Reb E”), rebaudioside D (“Reb D”)and rebaudioside M (“Reb M) standards. Enzymatic reaction by UGT76G1alone at 3 hr (D), 12 hr (G) and 24 hr (J); enzymatic reaction by theUGT-SUS (UGT76G1-AtSUS1) coupling system at 3 hr (E), 12 hr (H) and 24hr (K); enzymatic reaction by the GS fusion enzyme at 3 hr (F), 12 hr(I) and 24 hr (L).

FIG. 24 depicts the in vitro production of Reb D and Reb M fromstevioside catalyzed by a combination of a recombinant HV1, arecombinant UGT76G1, a GS fusion enzyme, and/or a recombinant AtSUS1.(A-D): showing the HPLC retention times of stevioside (“Ste”),rebaudioside A (“Reb A”), rebaudioside D (“Reb D”) and rebaudioside M(“Reb M) standards. Enzymatic reaction by HV1 and UGT76G1 in the UGT-SUScoupling system at 6 hr (E), 12 hr (H) and 24 hr (K); enzymatic reactionby HV1 and GS fusion enzyme at 6 hr (F), 12 hr (I) and 24 hr (L).Enzymatic reaction by UGT76G1 and HV1 at 6 hr (G), 12 hr (J) and 24 hr(M).

FIG. 25 depicts the in vitro production of Reb D and Reb M fromrebaudioside A catalyzed by a combination of recombinant HV1, arecombinant UGT76G1, a GS fusion enzyme, and/or a recombinant AtSUS1.(A-C): showing the HPLC retention times of rebaudioside A (“Reb A”),rebaudioside D (“Reb D”) and rebaudioside M (“Reb M) standards.Enzymatic reaction by HV1 and UGT76G1 in the UGT-SUS coupling system at6 hr (D), 12 hr (G) and 24 hr (J); enzymatic reaction by HV1 and GSfusion enzyme at 6 hr (E), 12 hr (H) and 24 hr (K). Enzymatic reactionby UGT76G1 and HV1 at 6 hr (F), 12 hr (I) and 24 hr (J).

FIG. 26 depicts the structure of Reb M.

FIG. 27 depicts the key TOCSY and HMBC correlations of Reb M.

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described below in detail. Itshould be understood, however, that the description of specificembodiments is not intended to limit the disclosure to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein may be usedin the practice or testing of the present disclosure, the preferredmaterials and methods are described below.

The term “complementary” is used according to its ordinary and customarymeaning as understood by a person of ordinary skill in the art, and isused without limitation to describe the relationship between nucleotidebases that are capable to hybridizing to one another. For example, withrespect to DNA, adenosine is complementary to thymine and cytosine iscomplementary to guanine. Accordingly, the subject technology alsoincludes isolated nucleic acid fragments that are complementary to thecomplete sequences as reported in the accompanying Sequence Listing aswell as those substantially similar nucleic acid sequences.

The terms “nucleic acid” and “nucleotide” are used according to theirrespective ordinary and customary meanings as understood by a person ofordinary skill in the art, and are used without limitation to refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar tonaturally-occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified or degenerate variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated.

The term “isolated” is used according to its ordinary and customarymeaning as understood by a person of ordinary skill in the art, and whenused in the context of an isolated nucleic acid or an isolatedpolypeptide, is used without limitation to refer to a nucleic acid orpolypeptide that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatednucleic acid or polypeptide can exist in a purified form or can exist ina non-native environment such as, for example, in a transgenic hostcell.

The terms “incubating” and “incubation” as used herein refers to aprocess of mixing two or more chemical or biological entities (such as achemical compound and an enzyme) and allowing them to interact underconditions favorable for producing a steviol glycoside composition.

The term “degenerate variant” refers to a nucleic acid sequence having aresidue sequence that differs from a reference nucleic acid sequence byone or more degenerate codon substitutions. Degenerate codonsubstitutions can be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed base and/or deoxyinosine residues. A nucleic acid sequence and allof its degenerate variants will express the same amino acid orpolypeptide.

The terms “polypeptide,” “protein,” and “peptide” are used according totheir respective ordinary and customary meanings as understood by aperson of ordinary skill in the art; the three terms are sometimes usedinterchangeably, and are used without limitation to refer to a polymerof amino acids, or amino acid analogs, regardless of its size orfunction. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “polypeptide” as used herein refers to peptides, polypeptides, andproteins, unless otherwise noted. The terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein when referring to apolynucleotide product. Thus, exemplary polypeptides includepolynucleotide products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants, andanalogs of the foregoing.

The terms “polypeptide fragment” and “fragment,” when used in referenceto a reference polypeptide, are used according to their ordinary andcustomary meanings to a person of ordinary skill in the art, and areused without limitation to refer to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions canoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both.

The term “functional fragment” of a polypeptide or protein refers to apeptide fragment that is a portion of the full length polypeptide orprotein, and has substantially the same biological activity, or carriesout substantially the same function as the full length polypeptide orprotein (e.g., carrying out the same enzymatic reaction).

The terms “variant polypeptide,” “modified amino acid sequence” or“modified polypeptide,” which are used interchangeably, refer to anamino acid sequence that is different from the reference polypeptide byone or more amino acids, e.g., by one or more amino acid substitutions,deletions, and/or additions. In an aspect, a variant is a “functionalvariant” which retains some or all of the ability of the referencepolypeptide.

The term “functional variant” further includes conservativelysubstituted variants. The term “conservatively substituted variant”refers to a peptide having an amino acid sequence that differs from areference peptide by one or more conservative amino acid substitutions,and maintains some or all of the activity of the reference peptide. A“conservative amino acid substitution” is a substitution of an aminoacid residue with a functionally similar residue. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another; the substitution of one charged or polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between threonine and serine; the substitutionof one basic residue such as lysine or arginine for another; or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another; or the substitution of one aromatic residue, such asphenylalanine, tyrosine, or tryptophan for another. Such substitutionsare expected to have little or no effect on the apparent molecularweight or isoelectric point of the protein or polypeptide. The phrase“conservatively substituted variant” also includes peptides wherein aresidue is replaced with a chemically-derivatized residue, provided thatthe resulting peptide maintains some or all of the activity of thereference peptide as described herein.

The term “variant,” in connection with the polypeptides of the subjecttechnology, further includes a functionally active polypeptide having anamino acid sequence at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, and even 100% identical to the amino acid sequence ofa reference polypeptide.

The term “homologous” in all its grammatical forms and spellingvariations refers to the relationship between polynucleotides orpolypeptides that possess a “common evolutionary origin,” includingpolynucleotides or polypeptides from superfamilies and homologouspolynucleotides or proteins from different species (Reeck et al., Cell50:667, 1987). Such polynucleotides or polypeptides have sequencehomology, as reflected by their sequence similarity, whether in terms ofpercent identity or the presence of specific amino acids or motifs atconserved positions. For example, two homologous polypeptides can haveamino acid sequences that are at least 75%, at least 76%, at least 77%,at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, and even 100% identical.

“Percent (%) amino acid sequence identity” with respect to the variantpolypeptide sequences of the subject technology refers to the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues of a reference polypeptide after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity.

Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared. For example, the %amino acid sequence identity may be determined using the sequencecomparison program NCBI-BLAST2. The NCBI-BLAST2 sequence comparisonprogram may be downloaded from ncbi.nlm.nih.gov. NCBI BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask yes, strand=all,expected occurrences 10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62. In situations whereNCBI-BLAST2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows: 100 times the fraction X/Y where Xis the number of amino acid residues scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Aand B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

In this sense, techniques for determining amino acid sequence“similarity” are well known in the art. In general, “similarity” refersto the exact amino acid to amino acid comparison of two or morepolypeptides at the appropriate place, where amino acids are identicalor possess similar chemical and/or physical properties such as charge orhydrophobicity. A so-termed “percent similarity” may then be determinedbetween the compared polypeptide sequences. Techniques for determiningnucleic acid and amino acid sequence identity also are well known in theart and include determining the nucleotide sequence of the mRNA for thatgene (usually via a cDNA intermediate) and determining the amino acidsequence encoded therein, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity”, as can two or more amino acid sequences. The programsavailable in the Wisconsin Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.), for example,the GAP program, are capable of calculating both the identity betweentwo polynucleotides and the identity and similarity between twopolypeptide sequences, respectively. Other programs for calculatingidentity or similarity between sequences are known by those skilled inthe art.

An amino acid position “corresponding to” a reference position refers toa position that aligns with a reference sequence, as identified byaligning the amino acid sequences. Such alignments can be done by handor by using well-known sequence alignment programs such as ClustalW2,Blast 2, etc.

Unless specified otherwise, the percent identity of two polypeptide orpolynucleotide sequences refers to the percentage of identical aminoacid residues or nucleotides across the entire length of the shorter ofthe two sequences.

“Coding sequence” is used according to its ordinary and customarymeaning as understood by a person of ordinary skill in the art, and isused without limitation to refer to a DNA sequence that encodes for aspecific amino acid sequence.

“Suitable regulatory sequences” is used according to its ordinary andcustomary meaning as understood by a person of ordinary skill in theart, and is used without limitation to refer to nucleotide sequenceslocated upstream (5′ non-coding sequences), within, or downstream (3′non-coding sequences) of a coding sequence, and which influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences may include promoters,translation leader sequences, introns, and polyadenylation recognitionsequences.

“Promoter” is used according to its ordinary and customary meaning asunderstood by a person of ordinary skill in the art, and is used withoutlimitation to refer to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent cell types, or at different stages of development, or inresponse to different environmental conditions. Promoters, which cause agene to be expressed in most cell types at most times, are commonlyreferred to as “constitutive promoters.” It is further recognized thatsince in most cases the exact boundaries of regulatory sequences havenot been completely defined, DNA fragments of different lengths may haveidentical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein, is used according to its ordinaryand customary meaning as understood by a person of ordinary skill in theart, and is used without limitation to refer to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the subject technology. “Over-expression”refers to the production of a gene product in transgenic or recombinantorganisms that exceeds levels of production in normal or non-transformedorganisms.

“Transformation” is used according to its ordinary and customary meaningas understood by a person of ordinary skill in the art, and is usedwithout limitation to refer to the transfer of a polynucleotide into atarget cell. The transferred polynucleotide can be incorporated into thegenome or chromosomal DNA of a target cell, resulting in geneticallystable inheritance, or it can replicate independent of the hostchromosomal. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “transformed,” “transgenic,” and “recombinant,” when usedherein in connection with host cells, are used according to theirordinary and customary meanings as understood by a person of ordinaryskill in the art, and are used without limitation to refer to a cell ofa host organism, such as a plant or microbial cell, into which aheterologous nucleic acid molecule has been introduced. The nucleic acidmolecule can be stably integrated into the genome of the host cell, orthe nucleic acid molecule can be present as an extrachromosomalmolecule. Such an extrachromosomal molecule can be auto-replicating.Transformed cells, tissues, or subjects are understood to encompass notonly the end product of a transformation process, but also transgenicprogeny thereof.

The terms “recombinant,” “heterologous,” and “exogenous,” when usedherein in connection with polynucleotides, are used according to theirordinary and customary meanings as understood by a person of ordinaryskill in the art, and are used without limitation to refer to apolynucleotide (e.g., a DNA sequence or a gene) that originates from asource foreign to the particular host cell or, if from the same source,is modified from its original form. Thus, a heterologous gene in a hostcell includes a gene that is endogenous to the particular host cell buthas been modified through, for example, the use of site-directedmutagenesis or other recombinant techniques. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a position orform within the host cell in which the element is not ordinarily found.

Similarly, the terms “recombinant,” “heterologous,” and “exogenous,”when used herein in connection with a polypeptide or amino acidsequence, means a polypeptide or amino acid sequence that originatesfrom a source foreign to the particular host cell or, if from the samesource, is modified from its original form. Thus, recombinant DNAsegments can be expressed in a host cell to produce a recombinantpolypeptide.

The terms “plasmid,” “vector,” and “cassette” are used according totheir ordinary and customary meanings as understood by a person ofordinary skill in the art, and are used without limitation to refer toan extra chromosomal element often carrying genes which are not part ofthe central metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described, for example, by Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual, 2^(nd) ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M.L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. etal., In Current Protocols in Molecular Biology, published by GreenePublishing and Wiley-Interscience, 1987; the entireties of each of whichare hereby incorporated herein by reference to the extent they areconsistent herewith.

As used herein, “synthetic” or “organically synthesized” or “chemicallysynthesized” or “organically synthesizing” or “chemically synthesizing”or “organic synthesis” or “chemical synthesis” are used to refer topreparing the compounds through a series of chemical reactions; thisdoes not include extracting the compound, for example, from a naturalsource.

The term “orally consumable product” as used herein refers to anybeverage, food product, dietary supplement, nutraceutical,pharmaceutical composition, dental hygienic composition and cosmeticproduct which are contacted with the mouth of man or animal, includingsubstances that are taken into and subsequently ejected from the mouthand substances which are drunk, eaten, swallowed, or otherwise ingested;and that are safe for human or animal consumption when used in agenerally acceptable range of concentrations.

The term “food product” as used herein refers to fruits, vegetables,juices, meat products such as ham, bacon and sausage; egg products,fruit concentrates, gelatins and gelatin-like products such as jams,jellies, preserves, and the like; milk products such as ice cream, sourcream, yogurt, and sherbet; icings, syrups including molasses; corn,wheat, rye, soybean, oat, rice and barley products, cereal products, nutmeats and nut products, cakes, cookies, confectionaries such as candies,gums, fruit flavored drops, and chocolates, chewing gum, mints, creams,icing, ice cream, pies and breads. “Food product” also refers tocondiments such as herbs, spices and seasonings, flavor enhancers, suchas monosodium glutamate. “Food product” further refers to also includesprepared packaged products, such as dietetic sweeteners, liquidsweeteners, tabletop flavorings, granulated flavor mixes which uponreconstitution with water provide non-carbonated drinks, instant puddingmixes, instant coffee and tea, coffee whiteners, malted milk mixes, petfoods, livestock feed, tobacco, and materials for baking applications,such as powdered baking mixes for the preparation of breads, cookies,cakes, pancakes, donuts and the like. “Food product” also refers to dietor low-calorie food and beverages containing little or no sucrose.

As used herein, the term “stereoisomer” is a general term for allisomers of individual molecules that differ only in the orientation oftheir atoms in space. “Stereoisomer” includes enantiomers and isomers ofcompounds with more than one chiral center that are not mirror images ofone another (diastereomers).

As used herein, the term “amorphous rebaudioside V” refers to anon-crystalline solid form of rebaudioside V. As used herein, the term“amorphous rebaudioside W” refers to a non-crystalline solid form ofrebaudioside W.

As used herein, the term “sweetness intensity” refers to the relativestrength of sweet sensation as observed or experienced by an individual,e.g., a human, or a degree or amount of sweetness detected by a taster,for example on a Brix scale.

As used herein, the term “enhancing the sweetness” refers to the effectof rebaudioside V and/or rebaudioside W in increasing, augmenting,intensifying, accentuating, magnifying, and/or potentiating the sensoryperception of one or more sweetness characteristics of a beverageproduct or a consumable product of the present disclosure withoutchanging the nature or quality thereof, as compared to a correspondingorally consumable product that does not contain rebaudioside V and/orrebaudioside W.

As used herein, the term “off-taste(s)” refers to an amount or degree oftaste that is not characteristically or usually found in a beverageproduct or a consumable product of the present disclosure. For example,an off-taste is an undesirable taste of a sweetened consumable toconsumers, such as, a bitter taste, a licorice-like taste, a metallictaste, an aversive taste, an astringent taste, a delayed sweetnessonset, a lingering sweet aftertaste, and the like, etc.

As used herein, the term “w/v-%” refers to the weight of a compound,such as a sugar, (in grams) for every 100 ml of a liquid orallyconsumable product of the present disclosure containing such compound.As used herein, the term “w/w-%” refers to the weight of a compound,such as a sugar, (in grams) for every gram of an orally consumableproduct of the present disclosure containing such compound.

As used herein, the term “ppm” refers to part(s) per million by weight,for example, the weight of a compound, such as rebaudioside V and/orrebaudioside W (in milligrams) per kilogram of an orally consumableproduct of the present disclosure containing such compound (i.e., mg/kg)or the weight of a compound, such as rebaudioside V and/or rebaudiosideW (in milligrams) per liter of an orally consumable product of thepresent disclosure containing such compound (i.e., mg/L); or by volume,for example the volume of a compound, such as rebaudioside V and/orrebaudioside W (in milliliters) per liter of an orally consumableproduct of the present disclosure containing such compound (i.e., ml/L).

In accordance with the present disclosure, non-caloric sweeteners andmethods for synthesizing the non-caloric sweeteners are disclosed. Alsoin accordance with the present disclosure an enzyme and methods of usingthe enzyme to prepare the non-caloric sweeteners are disclosed.

Synthetic Non-Caloric Sweeteners: Synthetic Rebaudioside V

In one aspect, the present disclosure is directed to a syntheticnon-caloric sweetener. The synthetic non-caloric sweetener is asynthetic rebaudioside-type steviol glycoside and has been given thename, “Rebaudioside V”. Rebaudioside V (“Reb V”) is a steviol glycosidehaving four β-D-glucosyl units in its structure connected to theaglycone steviol, a steviol aglycone moiety with a Glc β1-3-Glc β1 unitat C-13 in the form of ether linkage and another Glc β1-2-Glc β1 unit atC-19 position in the form of an ester linkage.

Rebaudioside V has the molecular formula C₄₄H₇₀O₂₃ and the IUPAC name,13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester on the basis of extensive 1D and 2D NMR as well as high resolutionmass spectral data and hydrolysis studies.

Synthetic Non-Caloric Sweeteners: Synthetic Rebaudioside W

In one aspect, the present disclosure is directed to a syntheticnon-caloric sweetener. The synthetic non-caloric sweetener is asynthetic rebaudioside-type steviol glycoside and has been given thename, “Rebaudioside W”. Rebaudioside W (“Reb W”) is a steviol glycosidehaving five β-D-glucosyl units in its structure connected to theaglycone steviol, a steviol aglycone moiety with a Glc β1-3-Glc β1 unitat C-13 in the form of ether linkage and a Glc β1-2(Glc β1-3)-Glc β1unit at C-19 position in the form of an ester linkage.

Rebaudioside W has the molecular formula C₅₀H₈₀O₂₈ and the IUPAC name,13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

Synthetic Non-Caloric Sweeteners: Synthetic Rebaudioside KA

In one aspect, the present disclosure is directed to a syntheticnon-caloric sweetener. The synthetic non-caloric sweetener is asynthetic rebaudioside-type steviol glycoside and has been given thename, “Rebaudioside KA”. Rebaudioside KA (“Reb KA”) is a steviolglycoside having three β-D-glucosyl units in its structure connected tothe aglycone steviol, a steviol aglycone moiety with a Glc β1 unit atC-13 in the form of ether linkage and a Glc β1-2-Glc β1 unit at C-19 inthe form of ether linkage. Rebaudioside KA has the molecular formulaC₃₈H₆₀O₁₈ and the IUPAC name, 13-β-D-glucopyranosyloxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester on the basis of extensive 1D and 2D NMR as well as high resolutionmass spectral data and hydrolysis studies.

Synthetic Non-Caloric Sweeteners: Synthetic Rebaudioside G

In one aspect, the present disclosure is directed to a syntheticnon-caloric sweetener. The synthetic non-caloric sweetener is asynthetic rebaudioside-type steviol glycoside and has been given thename, “Rebaudioside G”. Rebaudioside G (“Reb G”) is a steviol glycosidehaving three β-D-glucosyl units in its structure connected to theaglycone steviol, a steviol aglycone moiety with a Glc β1-3-Glc β1 unitat C-13 in the form of ether linkage and a Glc β1 unit at C-19 in theform of ether linkage.

Rebaudioside G has the molecular formula C₃₈H₆₀O₁₈ and the IUPAC name,13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl) ester on the basis ofextensive 1D and 2D NMR as well as high resolution mass spectral dataand hydrolysis studies.

Synthetic Non-Caloric Sweeteners: Synthetic Rebaudioside M

In one aspect, the present disclosure is directed to a syntheticnon-caloric sweetener. The synthetic non-caloric sweetener is asynthetic rebaudioside-type steviol glycoside and has been given thename, “Rebaudioside M”. Rebaudioside M (“Reb M”) is a steviol glycosidehaving six β-D-glucosyl units in its structure connected to the aglyconesteviol, a steviol aglycone moiety with a Glc β1-2(Glc β1-3)-Glc β1 unitat the C-13 position in the form of an ether linkage and a Glc β1-2(Glcβ1-3)-Glc β1 unit at the C-19 position in the form of an ester linkage.

Rebaudioside M has the molecular formula C₅₆H₉₀O₃₃ and the IUPAC name,13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)esteron the basis of extensive 1D and 2D NMR as well as high resolution massspectral data and hydrolysis studies.

Methods of Synthesizing Steviol Glycosides

Method of Producing Rebaudioside V from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rebaudioside G. The method comprisespreparing a reaction mixture comprising rebaudioside G; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); and a HV1UDP-glycosyltransferase; with or without sucrose synthase (SUS) andincubating the reaction mixture for a sufficient time to producerebaudioside V, wherein a glucose is covalently coupled to therebaudioside G to produce rebaudioside V.

Method of Producing Rebaudioside V from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rebaudioside G. The method comprisespreparing a reaction mixture comprising rebaudioside G; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); a uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of an EUGT11, a UDP-glycosyltransferase-Sucrose synthase(SUS) fusion enzyme; with or without sucrose synthase (SUS) andincubating the reaction mixture for a sufficient time to producerebaudioside V, wherein a glucose is covalently coupled to therebaudioside G to produce rebaudioside V.

Method of producing Rebaudioside V from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rebaudioside KA. The method comprisespreparing a reaction mixture comprising rebaudioside KA; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); a uridine diposphoglycosyltransferases (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1) and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; with or withoutsucrose synthase (SUS) and incubating the reaction mixture for asufficient time to produce rebaudioside V, wherein a glucose iscovalently coupled to the rebaudioside KA to produce rebaudioside V.

Method of Producing Rebaudioside V from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside V from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); uridine diposphoglycosyltransferase(s) (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1), HV1 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; with or withoutsucrose synthase (SUS) and incubating the reaction mixture for asufficient time to produce rebaudioside V, wherein a glucose iscovalently coupled to the rubusoside to produce rebaudioside KA.Continually, a glucose is covalently coupled to the rebaudioside KA toproduce rebaudioside V.

Method of Producing of Rebaudioside V from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a mixture of rebaudioside A and rebaudioside V fromrubusoside. The method comprises preparing a reaction mixture comprisingrubusoside; substrates selected from the group consisting of sucrose,uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose);uridine dipospho glycosyltransferase(s) (UDP-glycosyltransferase)selected from the group consisting of a UDP-glycosyltransferase(UGT76G1), EUGT11 and a UDP-glycosyltransferase-Sucrose synthase fusionenzyme; with or without sucrose synthase; and incubating the reactionmixture for a sufficient time to produce rebaudioside V, wherein aglucose is covalently coupled to the rubusoside to produce rebaudiosideKA and a glucose is covalently coupled to the rebaudioside KA to producerebaudioside V. A glucose is covalently coupled to the rubusoside toproduce rebaudioside G. Continually, a glucose is covalently coupled tothe rebaudioside G to produce rebaudioside V.

Method of producing Rebaudioside W from Rebaudioside V.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside V. The method comprisespreparing a reaction mixture comprising rebaudioside V; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); an uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a UDP-glycosyltransferase (UGT76G1) and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; with or withoutsucrose synthase and incubating the reaction mixture for a sufficienttime to produce rebaudioside W, wherein a glucose is covalently coupledto the rebaudioside V to produce rebaudioside W.

Method of Producing Rebaudioside W from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside G. The method comprisespreparing a reaction mixture comprising rebaudioside G; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); an uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a uridine diphospho glycosyltransferase (UGT76G1), aUDP-glycosyltransferase-Sucrose synthase fusion enzyme and a HV1; withor without sucrose synthase; and incubating the reaction mixture for asufficient time to produce rebaudioside W, wherein a glucose iscovalently coupled to the rebaudioside G to produce rebaudioside V byHV1. Continually, a glucose is covalently coupled to the rebaudioside Vto produce rebaudioside W by UGT76G1.

Method of Producing Rebaudioside W from Rebaudioside G.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside G. The method comprisespreparing a reaction mixture comprising rebaudioside G; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); an uridine diphosphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a UGT76G1, an EUGT11, and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; and incubatingthe reaction mixture for a sufficient time to produce rebaudioside W,wherein a glucose is covalently coupled to the rebaudioside G to producerebaudioside V by EUGT11. Continually, a glucose is covalently coupledto the rebaudioside V to produce rebaudioside W by UGT76G1.

Method of Producing Rebaudioside W from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rebaudioside KA. The method comprisespreparing a reaction mixture comprising rebaudioside KA; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); an uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a uridine diphospho glycosyltransferase (UGT76G1), and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside W, wherein a glucose is covalently coupledto the rebaudioside KA to produce rebaudioside V. Continually, a glucoseis covalently coupled to the rebaudioside V to produce rebaudioside W.

Method of Producing of Rebaudioside W from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); uridine diphosphoglycosyltransferases selected from the group consisting of a UGT76G1, anHV1, and a UDP-glycosyltransferase-Sucrose synthase fusion enzyme; withor without sucrose synthase and incubating the reaction mixture for asufficient time to produce a mixture of rebaudioside W.

Method of Producing of Rebaudioside W from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside W from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); uridine diphosphoglycosyltransferases selected from the group consisting of a UGT76G1, anEUGT11, and a UDP-glycosyltransferase-Sucrose synthase fusion enzyme;with or without sucrose synthase and incubating the reaction mixture fora sufficient time to produce rebaudioside W.

Method of Producing a Mixture of Stevioside and Rebaudioside KA fromRubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a mixture of stevioside and rebaudioside KA fromrubusoside. The method comprises preparing a reaction mixture comprisingrubusoside; substrates selected from the group consisting of sucrose,uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose);a UDP-glycosyltransferase selected from the group consisting of EUGT11and a UDP-glycosyltransferase-Sucrose synthase fusion enzyme; with orwithout sucrose synthase; and incubating the reaction mixture for asufficient time to produce a mixture of stevioside and rebaudioside KA,wherein a glucose is covalently coupled to C2′-19-O-glucose ofrubusoside to produce rebaudioside KA; a glucose is convalently coupledto C2′-13-O-glucose of rubusoside to produce stevioside.

Method of Producing Rebaudioside KA from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a rebaudioside KA from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); and HV1UDP-glycosyltransferase; with or without sucrose synthase; andincubating the reaction mixture for a sufficient time to producerebaudioside KA, wherein a glucose is covalently coupled to theC2′-19-O-glucose of rubusoside to produce a rebaudioside KA.

Method of Producing Rebaudioside G from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing a rebaudioside G from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferaseselected from the group consisting of UGT76G1 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme; with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside G, wherein a glucose is covalently coupledto the C3′-13-O-glucose of rubusoside to produce a rebaudioside G.

Method of Producing Rebaudioside E from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rebaudioside KA. The method comprisespreparing a reaction mixture comprising rebaudioside KA; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); and HV1UDP-glycosyltransferase; with or without sucrose synthase; andincubating the reaction mixture for a sufficient time to producerebaudioside E, wherein a glucose is covalently coupled to the C2′13-O-glucose of rebaudioside KA to produce rebaudioside E.

Method of Producing Rebaudioside E from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rebaudioside KA. The method comprisespreparing a reaction mixture comprising rebaudioside KA; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferasefrom a group of EUGT11 and a UDP-glycosyltransferase-Sucrose synthasefusion enzyme; with or without sucrose synthase; and incubating thereaction mixture for a sufficient time to produce rebaudioside E,wherein a glucose is covalently coupled to the C2′ 13-O-glucose ofrebaudioside KA to produce rebaudioside E.

Method of Producing Rebaudioside E from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; a substrate selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); and a UDP-glycosyltransferasefrom the group of EUGT11 and a UDP-glycosyltransferase-Sucrose synthesisfusion enzyme; with or without sucrose synthase; incubating the reactionmixture for a sufficient time to produce rebaudioside E, wherein aglucose is covalently coupled to rubusoside to produce a mixture ofrebaudioside KA and stevioside. Continually, a glucose is covalentlycoupled to rebaudioside KA and stevioside to produce rebaudioside E.

Method of Producing Rebaudioside E from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); and HV1UDP-glycosyltransferase; with or without sucrose synthase; incubatingthe reaction mixture for a sufficient time to produce rebaudioside E,wherein a glucose is covalently coupled to the rubusoside to producerebaudioside KA; and further incubating the rebaudioside KA with HV1 toproduce rebaudioside E.

Method of Producing Rebaudioside D3 from Rubusoside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D3 from rubusoside. The method comprisespreparing a reaction mixture comprising rubusoside; substrates selectedfrom the group consisting of sucrose, uridine diphosphate (UDP) anduridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferasefrom the group of EUGT11 and a UDP-glycosyltransferase-Sucrose synthesisfusion enzyme; with or without sucrose synthase; incubating the reactionmixture for a sufficient time to produce rebaudioside D3, wherein aglucose is covalently coupled to the rubusoside to produce a mixture ofstevioside and rebaudioside KA; further incubating the mixture ofstevioside and rebaudioside KA with EUGT11 to produce rebaudioside E,wherein a glucose is covalently coupled to the stevioside and therebaudioside KA to produce rebaudioside E; and further incubating therebaudioside E with EUGT11 to produce rebaudioside D3, wherein a glucoseis covalently coupled to the rebaudioside E to produce rebaudioside D3.

Method of Producing Rebaudioside D3 from Rebaudioside KA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D3 from rebaudioside KA. The method includespreparing a reaction mixture comprising rebaudioside KA, substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferaseselected from the group consisting of an EUGT11 and aUDP-glycosyltransferase-Sucrose synthase fusion enzyme, with or withoutsucrose synthase; incubating the reaction mixture for a sufficient timeto produce rebaudioside D3, wherein a glucose is covalently coupled tothe rebaudioside KA to produce rebaudioside E; further incubating themixture of rebaudioside E with EUGT11 to produce rebaudioside D3,wherein a glucose is covalently coupled to the rebaudioside E to producerebaudioside D3.

Method of Producing Rebaudioside Z from Rebaudioside E.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside Z from rebaudioside E. The method comprisespreparing a reaction mixture comprising rebaudioside E; substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); and HV1UDP-glycosyltransferase; and sucrose synthase, incubating the reactionmixture for a sufficient time to produce rebaudioside Z, wherein aglucose is covalently coupled to the rebaudioside E to producerebaudioside Z, wherein a glucose is covalently coupled to theC2′-13-O-glucose of rebaudioside E to produce rebaudioside Z1. A glucoseis convalently coupled to C2′-19-O-glucose of rebaudioside E to producerebaudioside Z2.

Method of Producing Rebaudioside M from Rebaudioside D.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside M from rebaudioside D. The method includespreparing a reaction mixture comprising rebaudioside D, substratesselected from the group consisting of sucrose, uridine diphosphate(UDP), uridine diphosphate-glucose (UDP-glucose), and combinationsthereof, and a UDP-glycosyltransferase selected from the groupconsisting of UGT76G1, a UDP-glycosyltransferase-Sucrose synthase fusionenzyme, and combinations thereof, with or without sucrose synthase; andincubating the reaction mixture for a sufficient time to producerebaudioside M, wherein a glucose is covalently coupled to therebaudioside D to produce rebaudioside M.

Method of Producing Rebaudioside D and Rebaudioside M from Stevioside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D and rebaudioside M from stevioside. Themethod includes preparing a reaction mixture comprising stevioside,substrates selected from the group consisting of sucrose, uridinediphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), andcombinations thereof, and a UDP-glycosyltransferase selected from thegroup consisting of HV1, UGT76G1, a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, and combinations thereof, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside D and/or rebaudioside M. For instance, inembodiments, the reaction mixture may be incubated for a sufficient timeto produce rebaudioside D, and the reaction mixture comprisingrebaudioside D further incubated (e.g., with UGT76G1 and/or the fusionenzyme) to produce rebaudioside M. In certain embodiments, the reactionmixture will comprise HV1 and UGT76G1. In other embodiments, thereaction mixture will comprise HV1 and the fusion enzyme.

In certain embodiments, a glucose is covalently coupled to thestevioside to produce rebaudioside A and/or rebaudioside E. For example,a glucose may be covalently coupled to the stevioside by UGT76G1 or thefusion enzyme to produce rebaudioside A and/or a glucose may becovalently coupled to the stevioside by HV1 to produce rebaudioside E.Continually, a glucose may be covalently coupled to the rebaudioside Aby HV1 to produce rebaudioside D and/or a glucose may be covalentlycoupled to the rebaudioside E by UGT76G1 or the fusion enzyme to producerebaudioside D. A glucose may further be covalently coupled to therebaudioside D by UGT76G1 or the fusion enzyme to produce rebaudiosideM.

Method of Producing Rebaudioside D and Rebaudioside M from RebaudiosideA.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D and rebaudioside M from rebaudioside A. Themethod includes preparing a reaction mixture comprising rebaudioside A,substrates selected from the group consisting of sucrose, uridinediphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), andcombinations thereof, and a UDP-glycosyltransferase selected from thegroup consisting of HV1, UGT76G1, a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, and combinations thereof, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside D and/or rebaudioside M. For instance, inembodiments, the reaction mixture (e.g., comprising HV1) may beincubated for a sufficient time to produce rebaudioside D, and thereaction mixture comprising rebaudioside D further incubated (e.g., withUGT76G1 and/or the fusion enzyme) to produce rebaudioside M. In certainembodiments, the reaction mixture will comprise HV1 and UGT76G1. Inother embodiments, the reaction mixture will comprise HV1 and the fusionenzyme.

A glucose is covalently coupled to the rebaudioside A to producerebaudioside D. For example, a glucose may be covalently coupled to therebaudioside A by HV1 to produce rebaudioside D. Continually, a glucosemay be covalently coupled to the rebaudioside D by UGT76G1 or the fusionenzyme to produce rebaudioside M.

Method of Producing Rebaudioside D and Rebaudioside M from RebaudiosideE.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D and rebaudioside M from rebaudioside E. Themethod includes preparing a reaction mixture comprising rebaudioside E,substrates selected from the group consisting of sucrose, uridinediphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), andcombinations thereof, and a UDP-glycosyltransferase selected from thegroup consisting of an UGT76G1, a UDP-glycosyltransferase-Sucrosesynthase fusion enzyme, and combinations thereof, with or withoutsucrose synthase; and incubating the reaction mixture for a sufficienttime to produce rebaudioside D and/or rebaudioside M. For instance, inembodiments, the reaction mixture (e.g., comprising UGT76G1 and/or thefusion enzyme) may be incubated for a sufficient time to producerebaudioside D, and the reaction mixture comprising rebaudioside Dfurther incubated to produce rebaudioside M.

A glucose is covalently coupled to the rebaudioside E to producerebaudioside D. For example, a glucose may be covalently coupled to therebaudioside E by UGT76G1 or the fusion enzyme to produce rebaudiosideD. Continually, a glucose may be covalently coupled to the rebaudiosideD by UGT76G1 or the fusion enzyme to produce rebaudioside M.

The majority of the steviol glycosides are formed by severalglycosylation reactions of steviol, which typically are catalyzed by theUDP-glycosyltransferases (UGTs) using uridine 5′-diphosphoglucose(UDP-glucose) as a donor of the sugar moiety. In plants, UGTs are a verydivergent group of enzymes that transfer a glucose residue fromUDP-glucose to steviol.

Uridine diphospho glycosyltransferase (UGT76G1) is a UGT with a1,3-13-O-glucose glycosylation activity to produce related glycoside(rebaudioside A and D). Surprisingly and unexpectedly, it was discoveredthat UGT76G1 also has 1,3-19-O-glucose glycosylation activity to producerebaudioside G from rubusoside, and to produce rebaudioside M fromrebaudioside D. UGT76G1 can convert rebaudioside KA to Reb V andcontinue to form Reb W. A particularly suitable UGT76G1 has an aminoacid sequence of SEQ ID NO:1.

EUGT11 (described in WO 2013022989) is a UGT having 1,2-19-O-glucose and1,2-13-O-glucose glycosylation activity. EUGT11 is known to catalyze theproduction of stevioside to rebaudioside E and rebaudioside A torebaudioside D. Surprisingly and unexpectedly, it was discovered thatEUGT11 can be used in vitro to synthesize rebaudioside D3 fromrebaudioside E by a new enzyme activity (β1,6-13-O-glucose glycosylationactivity) (U.S. patent application Ser. No. 14/269,435, assigned toConagen, Inc.). EUGT11 has 1,2-19-O-glucose glycosylation activity toproduce rebaudioside KA from rubusoside. A particularly suitable EUGT11has the amino acid sequence of SEQ ID NO:3.

HV1 is a UGT with a 1,2-19-O-glucose glycosylation activity to producerelated steviol glycosides (rebaudioside E, D and Z). Surprisingly andunexpectedly, it was discovered that HV1 also has 1,2-19-O-glucoseglycosylation activity to produce rebaudioside KA from rubusoside. HV1also can convert Reb G to Reb V and Reb KA to Reb E. A particularlysuitable HV1 has the amino acid sequence of SEQ ID NO:5.

The method can further include adding a sucrose synthase to the reactionmixture that contains the uridine diphospho (UDP) glycosyltransferase.Sucrose synthase catalyzes the chemical reaction between NDP-glucose andD-fructose to produce NDP and sucrose. Sucrose synthase is aglycosyltransferase. The systematic name of this enzyme class isNDP-glucose:D-fructose 2-alpha-D-glucosyltransferase. Other names incommon use include UDP glucose-fructose glucosyltransferase, sucrosesynthetase, sucrose-UDP glucosyltransferase, sucrose-uridine diphosphateglucosyltransferase, and uridine diphosphoglucose-fructoseglucosyltransferase. Addition of the sucrose synthase to the reactionmixture that includes a uridine diphospho glycosyltransferase creates a“UGT-SUS coupling system”. In the UGT-SUS coupling system, UDP-glucosecan be regenerated from UDP and sucrose, which allows for omitting theaddition of extra UDP-glucose to the reaction mixture or using UDP inthe reaction mixture. Suitable sucrose synthases can be for example, anArabidopsis sucrose synthase 1; an Arabidopsis sucrose synthase 3; and aVigna radiate sucrose synthase. A particularly suitable sucrose synthasecan be, for example, Arabidopsis sucrose synthase 1. A particularlysuitable Arabidopsis sucrose synthase 1 is Arabidopsis thaliana sucrosesynthase 1 (AtSUS1), having the amino acid sequence of SEQ ID NO:7.

In another aspect, uridine dipospho glycosyltransferase fusion enzymecan be used in the methods. A particularly suitable uridine diposphoglycosyltransferase fusion enzyme can be a UGT-SUS1 fusion enzyme. TheUDP-glycosyltransferase can be a UDP-glycosyltransferase fusion enzymethat includes a uridine diphospho glycosyltransferase domain coupled toa sucrose synthase domain. In particular, the UDP-glycosyltransferasefusion enzyme includes a uridine diphospho glycosyltransferase domaincoupled to a sucrose synthase domain. Additionally, the UGT-SUS1 fusionenzyme has sucrose synthase activity, and thus, can regenerateUDP-glucose from UDP and sucrose. A particularly suitable UGT-SUS1fusion enzyme can be, for example, a UGT76G1-AtSUS1 fusion enzyme (namedas: “GS”) having the amino acid sequence of SEQ ID NO:9. Anotherparticularly suitable UGT-SUS1 fusion enzyme can be, for example, aEUGT11-SUS1 (named as: “EUS”) having the amino acid sequence of SEQ IDNO:11.

Suitable sucrose synthase domains can be for example, an Arabidopsissucrose synthase 1; an Arabidopsis sucrose synthase 3; and a Vignaradiate sucrose synthase. A particularly suitable sucrose synthasedomain can be, for example, Arabidopsis sucrose synthase 1. Aparticularly suitable Arabidopsis sucrose synthase 1 is Arabidopsisthaliana sucrose synthase 1 (AtSUS1), having the amino acid sequence ofSEQ ID NO:7.

The UGT76G1-AtSUS1 (“GS”) fusion enzyme can have a polypeptide sequencewith at least 70%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% and even 100% identical tothe amino acid sequence set forth in SEQ ID NO:9. Suitably, the aminoacid sequence of the UGT-AtSUS1 fusion enzyme has at least 80% identityto SEQ ID NO:9. More suitably, the amino acid sequence of the UGT-AtSUS1fusion enzyme has at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, and even 100% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:9.

The isolated nucleic acid can include a nucleotide sequence encoding apolypeptide of the UGT-AtSUS1 fusion enzyme having a nucleic acidsequence that has at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, and even 100% sequence homology to the nucleic acid sequence setforth in SEQ ID NO:10. Suitably, the isolated nucleic acid includes anucleotide sequence encoding a polypeptide of theUDP-glycosyltransferase fusion enzyme having an amino acid sequence thatis at least 80% sequence identity to the amino acid sequence set forthin SEQ ID NO:9. More suitably, the isolated nucleic acid includes anucleotide sequence encoding a polypeptide of theUDP-glycosyltransferase fusion enzyme having an amino acid sequence thathas at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, and even 100% sequence identity to the amino acidsequence set forth in SEQ ID NO:9. The isolated nucleic acid thusincludes those nucleotide sequences encoding functional fragments of SEQID NO:10, functional variants of SEQ ID NO:9, or other homologouspolypeptides that have, for example, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, andeven 100% sequence identity to SEQ ID NO:9. As known by those skilled inthe art, the nucleic acid sequence encoding the UDP-glycosyltransferasecan be codon optimized for expression in a suitable host organism suchas, for example, bacteria and yeast.

The EUGT11-SUS1 (“EUS”) fusion enzyme can have a polypeptide sequencewith at least 70%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% and even 100% identical tothe amino acid sequence set forth in SEQ ID NO:11. Suitably, the aminoacid sequence of the EUGT11-SUS1 fusion enzyme has at least 80% identityto SEQ ID NO:11. More suitably, the amino acid sequence of theEUGT11-SUS1 fusion enzyme has at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, and even 100% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:11.

The isolated nucleic acid can include a nucleotide sequence encoding apolypeptide of the EUGT11-SUS1 fusion enzyme having a nucleic acidsequence that has at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, and even 100% sequence homology to the nucleic acid sequence setforth in SEQ ID NO:12. Suitably, the isolated nucleic acid includes anucleotide sequence encoding a polypeptide of the EUGT11-SUS1 fusionenzyme having an amino acid sequence that is at least 80% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:11. Moresuitably, the isolated nucleic acid includes a nucleotide sequenceencoding a polypeptide of the EUGT11-SUS1 fusion enzyme having an aminoacid sequence that has at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, and even 100% sequence identityto the amino acid sequence set forth in SEQ ID NO:11. The isolatednucleic acid thus includes those nucleotide sequences encodingfunctional fragments of SEQ ID NO:11, functional variants of SEQ IDNO:11, or other homologous polypeptides that have, for example, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, and even 100% sequence identity to SEQ ID NO:11. Asknown by those skilled in the art, the nucleic acid sequence encodingthe EUGT11-SUS1 can be codon optimized for expression in a suitable hostorganism such as, for example, bacteria and yeast.

Orally Consumable Products

In another aspect, the present disclosure is directed to an orallyconsumable product having a sweetening amount of rebaudioside V,selected from the group consisting of a beverage product and aconsumable product. In another aspect, the present disclosure isdirected to an orally consumable product having a sweetening amount ofrebaudioside W, selected from the group consisting of a beverage productand a consumable product. In another aspect, the present disclosure isdirected to an orally consumable product having a sweetening amount ofrebaudioside KA, selected from the group consisting of a beverageproduct and a consumable product. In another aspect, the presentdisclosure is directed to an orally consumable product having asweetening amount of rebaudioside G, selected from the group consistingof a beverage product and a consumable product. In another aspect, thepresent disclosure is directed to an orally consumable product having asweetening amount of rebaudioside M, selected from the group consistingof a beverage product and a consumable product.

The orally consumable product can have a sweetness intensity equivalentto about 1% (w/v-%) to about 4% (w/v-%) sucrose solution.

The orally consumable product can have from about 5 ppm to about 100 ppmrebaudioside V. The orally consumable product can have from about 5 ppmto about 100 ppm rebaudioside W. The orally consumable product can havefrom about 5 ppm to about 100 ppm rebaudioside KA. The orally consumableproduct can have from about 5 ppm to about 100 ppm rebaudioside G. Theorally consumable product can have from about 5 ppm to about 100 ppmrebaudioside M.

The rebaudioside V can be the only sweetener in the orally consumableproduct. The rebaudioside W can be the only sweetener in the orallyconsumable product. The rebaudioside KA can be the only sweetener in theorally consumable product. The rebaudioside G can be the only sweetenerin the orally consumable product. The rebaudioside M can be the onlysweetener in the orally consumable product.

The orally consumable product can also have at least one additionalsweetener. The at least one additional sweetener can be a natural highintensity sweetener, for example. The additional sweetener can beselected from a stevia extract, a steviol glycoside, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside D3, rebaudioside E, rebaudioside F, dulcoside A,rubusoside, steviolbioside, sucrose, high fructose corn syrup, fructose,glucose, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol,sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharine,naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC),rubusoside, mogroside IV, siamenoside I, mogroside V, monatin,thaumatin, monellin, brazzein, L-alanine, glycine, Lo Han Guo,hernandulcin, phyllodulcin, trilobtain, and combinations thereof.

The orally consumable product can also have at least one additive. Theadditive can be, for example, a carbohydrate, a polyol, an amino acid orsalt thereof, a polyamino acid or salt thereof, a sugar acid or saltthereof, a nucleotide, an organic acid, an inorganic acid, an organicsalt, an organic acid salt, an organic base salt, an inorganic salt, abitter compound, a flavorant, a flavoring ingredient, an astringentcompound, a protein, a protein hydrolysate, a surfactant, an emulsifier,a flavonoids, an alcohol, a polymer, and combinations thereof.

In one aspect, the present disclosure is directed to a beverage productcomprising a sweetening amount of rebaudioside V. In one aspect, thepresent disclosure is directed to a beverage product comprising asweetening amount of rebaudioside W. In one aspect, the presentdisclosure is directed to a beverage product comprising a sweeteningamount of rebaudioside KA. In one aspect, the present disclosure isdirected to a beverage product comprising a sweetening amount ofrebaudioside G. In one aspect, the present disclosure is directed to abeverage product comprising a sweetening amount of rebaudioside M.

The beverage product can be, for example, a carbonated beverage productand a non-carbonated beverage product. The beverage product can also be,for example, a soft drink, a fountain beverage, a frozen beverage; aready-to-drink beverage; a frozen and ready-to-drink beverage, coffee,tea, a dairy beverage, a powdered soft drink, a liquid concentrate,flavored water, enhanced water, fruit juice, a fruit juice flavoreddrink, a sport drink, and an energy drink.

In some embodiments, a beverage product of the present disclosure caninclude one or more beverage ingredients such as, for example,acidulants, fruit juices and/or vegetable juices, pulp, etc.,flavorings, coloring, preservatives, vitamins, minerals, electrolytes,erythritol, tagatose, glycerine, and carbon dioxide. Such beverageproducts may be provided in any suitable form, such as a beverageconcentrate and a carbonated, ready-to-drink beverage.

In certain embodiments, beverage products of the present disclosure canhave any of numerous different specific formulations or constitutions.The formulation of a beverage product of the present disclosure can varyto a certain extent, depending upon such factors as the product'sintended market segment, its desired nutritional characteristics, flavorprofile, and the like. For example, in certain embodiments, it cangenerally be an option to add further ingredients to the formulation ofa particular beverage product. For example, additional (i.e., moreand/or other) sweeteners can be added, flavorings, electrolytes,vitamins, fruit juices or other fruit products, tastents, masking agentsand the like, flavor enhancers, and/or carbonation typically may beadded to any such formulations to vary the taste, mouthfeel, nutritionalcharacteristics, etc. In embodiments, the beverage product can be a colabeverage that contains water, about 5 ppm to about 100 ppm rebaudiosideV, an acidulant, and flavoring. In embodiments, the beverage product canbe a cola beverage that contains water, about 5 ppm to about 100 ppmrebaudioside W, an acidulant, and flavoring. In embodiments, thebeverage product can be a cola beverage that contains water, about 5 ppmto about 100 ppm rebaudioside M, an acidulant, and flavoring. Exemplaryflavorings can be, for example, cola flavoring, citrus flavoring, andspice flavorings. In some embodiments, carbonation in the form of carbondioxide can be added for effervescence. In other embodiments,preservatives can be added, depending upon the other ingredients,production technique, desired shelf life, etc. In certain embodiments,caffeine can be added. In some embodiments, the beverage product can bea cola-flavored carbonated beverage, characteristically containingcarbonated water, sweetener, kola nut extract and/or other flavoring,caramel coloring, one or more acids, and optionally other ingredients.

Suitable amounts of rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G present in the beverage product canbe, for example, from about 5 ppm to about 100 ppm. In some embodiments,low concentrations of rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G, for example, less than 100 ppm, andhas an equivalent sweetness to sucrose solutions having concentrationsbetween 10,000 ppm to 30,000 ppm. The final concentration that rangesfrom about 5 ppm to about 100 ppm, from about 5 ppm to about 95 ppm,from about 5 ppm to about 90 ppm, from about 5 ppm to about 85 ppm, fromabout 5 ppm to about 80 ppm, from about 5 ppm to about 75 ppm, fromabout 5 ppm to about 70 ppm, from about 5 ppm to about 65 ppm, fromabout 5 ppm to about 60 ppm, from about 5 ppm to about 55 ppm, fromabout 5 ppm to about 50 ppm, from about 5 ppm to about 45 ppm, fromabout 5 ppm to about 40 ppm, from about 5 ppm to about 35 ppm, fromabout 5 ppm to about 30 ppm, from about 5 ppm to about 25 ppm, fromabout 5 ppm to about 20 ppm, from about 5 ppm to about 15 ppm, or fromabout 5 ppm to about 10 ppm. Alternatively, rebaudioside V orrebaudioside W can be present in beverage products of the presentdisclosure at a final concentration that ranges from about 5 ppm toabout 100 ppm, from about 10 ppm to about 100 ppm, from about 15 ppm toabout 100 ppm, from about 20 ppm to about 100 ppm, from about 25 ppm toabout 100 ppm, from about 30 ppm to about 100 ppm, from about 35 ppm toabout 100 ppm, from about 40 ppm to about 100 ppm, from about 45 ppm toabout 100 ppm, from about 50 ppm to about 100 ppm, from about 55 ppm toabout 100 ppm, from about 60 ppm to about 100 ppm, from about 65 ppm toabout 100 ppm, from about 70 ppm to about 100 ppm, from about 75 ppm toabout 100 ppm, from about 80 ppm to about 100 ppm, from about 85 ppm toabout 100 ppm, from about 90 ppm to about 100 ppm, or from about 95 ppmto about 100 ppm.

In another aspect, the present disclosure is directed to a consumablecomprising a sweetening amount of rebaudioside V. In another aspect, thepresent disclosure is directed to a consumable comprising a sweeteningamount of rebaudioside W. In another aspect, the present disclosure isdirected to a consumable comprising a sweetening amount of rebaudiosideKA. In another aspect, the present disclosure is directed to aconsumable comprising a sweetening amount of rebaudioside G. In anotheraspect, the present disclosure is directed to a consumable comprising asweetening amount of rebaudioside M. The consumable can be, for example,a food product, a nutraceutical, a pharmaceutical, a dietary supplement,a dental hygienic composition, an edible gel composition, a cosmeticproduct and a tabletop flavoring.

As used herein, “dietary supplement(s)” refers to compounds intended tosupplement the diet and provide nutrients, such as vitamins, minerals,fiber, fatty acids, amino acids, etc. that may be missing or may not beconsumed in sufficient quantities in a diet. Any suitable dietarysupplement known in the art may be used. Examples of suitable dietarysupplements can be, for example, nutrients, vitamins, minerals, fiber,fatty acids, herbs, botanicals, amino acids, and metabolites.

As used herein, “nutraceutical(s)” refers to compounds, which includesany food or part of a food that may provide medicinal or healthbenefits, including the prevention and/or treatment of disease ordisorder (e.g., fatigue, insomnia, effects of aging, memory loss, mooddisorders, cardiovascular disease and high levels of cholesterol in theblood, diabetes, osteoporosis, inflammation, autoimmune disorders,etc.). Any suitable nutraceutical known in the art may be used. In someembodiments, nutraceuticals can be used as supplements to food andbeverages and as pharmaceutical formulations for enteral or parenteralapplications which may be solid formulations, such as capsules ortablets, or liquid formulations, such as solutions or suspensions.

In some embodiments, dietary supplements and nutraceuticals can furthercontain protective hydrocolloids (such as gums, proteins, modifiedstarches), binders, film-forming agents, encapsulating agents/materials,wall/shell materials, matrix compounds, coatings, emulsifiers, surfaceactive agents, solubilizing agents (oils, fats, waxes, lecithins, etc.),adsorbents, carriers, fillers, co-compounds, dispersing agents, wettingagents, processing aids (solvents), flowing agents, taste-maskingagents, weighting agents, jellyfying agents, gel-forming agents,antioxidants and antimicrobials.

As used herein, a “gel” refers to a colloidal system in which a networkof particles spans the volume of a liquid medium. Although gels mainlyare composed of liquids, and thus exhibit densities similar to liquids,gels have the structural coherence of solids due to the network ofparticles that spans the liquid medium. For this reason, gels generallyappear to be solid, jelly-like materials. Gels can be used in a numberof applications. For example, gels can be used in foods, paints, andadhesives. Gels that can be eaten are referred to as “edible gelcompositions.” Edible gel compositions typically are eaten as snacks, asdesserts, as a part of staple foods, or along with staple foods.Examples of suitable edible gel compositions can be, for example, geldesserts, puddings, jams, jellies, pastes, trifles, aspics,marshmallows, gummy candies, and the like. In some embodiments, ediblegel mixes generally are powdered or granular solids to which a fluid maybe added to form an edible gel composition. Examples of suitable fluidscan be, for example, water, dairy fluids, dairy analogue fluids, juices,alcohol, alcoholic beverages, and combinations thereof. Examples ofsuitable dairy fluids can be, for example, milk, cultured milk, cream,fluid whey, and mixtures thereof. Examples of suitable dairy analoguefluids can be, for example, soy milk and non-dairy coffee whitener.

As used herein, the term “gelling ingredient” refers to any materialthat can form a colloidal system within a liquid medium. Examples ofsuitable gelling ingredients can be, for example, gelatin, alginate,carageenan, gum, pectin, konjac, agar, food acid, rennet, starch, starchderivatives, and combinations thereof. It is well known to those in theart that the amount of gelling ingredient used in an edible gel mix oran edible gel composition can vary considerably depending on a number offactors such as, for example, the particular gelling ingredient used,the particular fluid base used, and the desired properties of the gel.

Gel mixes and gel compositions of the present disclosure can be preparedby any suitable method known in the art. In some embodiments, edible gelmixes and edible gel compositions of the present disclosure can beprepared using other ingredients in addition to the gelling agent.Examples of other suitable ingredients can be, for example, a food acid,a salt of a food acid, a buffering system, a bulking agent, asequestrant, a cross-linking agent, one or more flavors, one or morecolors, and combinations thereof.

Any suitable pharmaceutical composition known in the art may be used. Incertain embodiments, a pharmaceutical composition of the presentdisclosure can contain from about 5 ppm to about 100 ppm of rebaudiosideV, and one or more pharmaceutically acceptable excipients. In certainembodiments, a pharmaceutical composition of the present disclosure cancontain from about 5 ppm to about 100 ppm of rebaudioside W, and one ormore pharmaceutically acceptable excipients. In certain embodiments, apharmaceutical composition of the present disclosure can contain fromabout 5 ppm to about 100 ppm of rebaudioside KA, and one or morepharmaceutically acceptable excipients. In certain embodiments, apharmaceutical composition of the present disclosure can contain fromabout 5 ppm to about 100 ppm of rebaudioside G, and one or morepharmaceutically acceptable excipients. In certain embodiments, apharmaceutical composition of the present disclosure can contain fromabout 5 ppm to about 100 ppm of rebaudioside M, and one or morepharmaceutically acceptable excipients. In some embodiments,pharmaceutical compositions of the present disclosure can be used toformulate pharmaceutical drugs containing one or more active agents thatexert a biological effect. Accordingly, in some embodiments,pharmaceutical compositions of the present disclosure can contain one ormore active agents that exert a biological effect. Suitable activeagents are well known in the art (e.g., The Physician's Desk Reference).Such compositions can be prepared according to procedures well known inthe art, for example, as described in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., USA.

Rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, orrebaudioside G can be used with any suitable dental and oral hygienecompositions known in the art. Examples of suitable dental and oralhygiene compositions can be, for example, toothpastes, tooth polishes,dental floss, mouthwashes, mouth rinses, dentrifices, mouth sprays,mouth refreshers, plaque rinses, dental pain relievers, and the like.

Suitable amounts of rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G present in the consumable can be, forexample, from about 5 parts per million (ppm) to about 100 parts permillion (ppm). In some embodiments, low concentrations of rebaudiosideV, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G,for example, less than 100 ppm, has an equivalent sweetness to sucrosesolutions having concentrations between 10,000 ppm to 30,000 ppm. Thefinal concentration that ranges from about 5 ppm to about 100 ppm, fromabout 5 ppm to about 95 ppm, from about 5 ppm to about 90 ppm, fromabout 5 ppm to about 85 ppm, from about 5 ppm to about 80 ppm, fromabout 5 ppm to about 75 ppm, from about 5 ppm to about 70 ppm, fromabout 5 ppm to about 65 ppm, from about 5 ppm to about 60 ppm, fromabout 5 ppm to about 55 ppm, from about 5 ppm to about 50 ppm, fromabout 5 ppm to about 45 ppm, from about 5 ppm to about 40 ppm, fromabout 5 ppm to about 35 ppm, from about 5 ppm to about 30 ppm, fromabout 5 ppm to about 25 ppm, from about 5 ppm to about 20 ppm, fromabout 5 ppm to about 15 ppm, or from about 5 ppm to about 10 ppm.Alternatively, rebaudioside V or rebaudioside W can be present inconsumable products of the present disclosure at a final concentrationthat ranges from about 5 ppm to about 100 ppm, from about 10 ppm toabout 100 ppm, from about 15 ppm to about 100 ppm, from about 20 ppm toabout 100 ppm, from about 25 ppm to about 100 ppm, from about 30 ppm toabout 100 ppm, from about 35 ppm to about 100 ppm, from about 40 ppm toabout 100 ppm, from about 45 ppm to about 100 ppm, from about 50 ppm toabout 100 ppm, from about 55 ppm to about 100 ppm, from about 60 ppm toabout 100 ppm, from about 65 ppm to about 100 ppm, from about 70 ppm toabout 100 ppm, from about 75 ppm to about 100 ppm, from about 80 ppm toabout 100 ppm, from about 85 ppm to about 100 ppm, from about 90 ppm toabout 100 ppm, or from about 95 ppm to about 100 ppm.

In certain embodiments, from about 5 ppm to about 100 ppm ofrebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, orrebaudioside G is present in food product compositions. As used herein,“food product composition(s)” refers to any solid or liquid ingestiblematerial that can, but need not, have a nutritional value and beintended for consumption by humans and animals.

Examples of suitable food product compositions can be, for example,confectionary compositions, such as candies, mints, fruit flavoreddrops, cocoa products, chocolates, and the like; condiments, such asketchup, mustard, mayonnaise, and the like; chewing gums; cerealcompositions; baked goods, such as breads, cakes, pies, cookies, and thelike; dairy products, such as milk, cheese, cream, ice cream, sourcream, yogurt, sherbet, and the like; tabletop sweetener compositions;soups; stews; convenience foods; meats, such as ham, bacon, sausages,jerky, and the like; gelatins and gelatin-like products such as jams,jellies, preserves, and the like; fruits; vegetables; egg products;icings; syrups including molasses; snacks; nut meats and nut products;and animal feed.

Food product compositions can also be herbs, spices and seasonings,natural and synthetic flavors, and flavor enhancers, such as monosodiumglutamate. In some embodiments, food product compositions can be, forexample, prepared packaged products, such as dietetic sweeteners, liquidsweeteners, granulated flavor mixes, pet foods, livestock feed, tobacco,and materials for baking applications, such as powdered baking mixes forthe preparation of breads, cookies, cakes, pancakes, donuts and thelike. In other embodiments, food product compositions can also be dietand low-calorie food and beverages containing little or no sucrose.

In certain embodiments that may be combined with any of the precedingembodiments, the rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G is the only sweetener, and the producthas a sweetness intensity equivalent to about 1% to about 4% (w/v-%)sucrose solution. In certain embodiments that can be combined with anyof the preceding embodiments, the consumable products and beverageproducts can further include an additional sweetener, where the producthas a sweetness intensity equivalent to about 1% to about 10% (w/v-%)sucrose solution. In certain embodiments that can be combined with anyof the preceding embodiments, every sweetening ingredient in the productis a high intensity sweetener. In certain embodiments that can becombined with any of the preceding embodiments, every sweeteningingredient in the product can a natural high intensity sweetener. Incertain embodiments that can be combined with any of the precedingembodiments, the additional sweetener contains one or more sweetenersselected from a stevia extract, a steviol glycoside, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside D3, rebaudioside F, dulcoside A, rubusoside,steviolbioside, sucrose, high fructose corn syrup, fructose, glucose,xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol,inositol, AceK, aspartame, neotame, sucralose, saccharine, naringindihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC),rubusoside mogroside IV, siamenoside I, mogroside V, monatin, thaumatin,monellin, brazzein, L-alanine, glycine, Lo Han Guo, hernandulcin,phyllodulcin, trilobtain, and combinations thereof. In certainembodiments that can be combined with any of the preceding embodiments,the consumable products and beverage products can further include one ormore additives selected from a carbohydrate, a polyol, an amino acid orsalt thereof, a poly-amino acid or salt thereof, a sugar acid or saltthereof, a nucleotide, an organic acid, an inorganic acid, an organicsalt, an organic acid salt, an organic base salt, an inorganic salt, abitter compound, a flavorant, a flavoring ingredient, an astringentcompound, a protein, a protein hydrolysate, a surfactant, an emulsifier,a flavonoids, an alcohol, a polymer, and combinations thereof. Incertain embodiments that can be combined with any of the precedingembodiments, the rebaudioside D3 has a purity of about 50% to about 100%by weight before it is added into the product.

Sweetener

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In certain embodiments, the sweetener can further include at least oneof a filler, a bulking agent and an anticaking agent. Suitable fillers,bulking agents and anticaking agents are known in the art.

In certain embodiments, rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G sweetener can be included and/or addedat a final concentration that is sufficient to sweeten and/or enhancethe sweetness of the consumable products and beverage products. The“final concentration” of rebaudioside V, rebaudioside W, rebaudiosideKA, rebaudioside M, or rebaudioside G refers to the concentration ofrebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, orrebaudioside G present in the final consumable products and beverageproducts (i.e., after all ingredients and/or compounds have been addedto produce the consumable products and beverage products). Accordingly,in certain embodiments, rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G is included and/or added to a compoundor ingredient used to prepare the consumable products and beverageproducts. The rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G may be present in a single compound oringredient, or multiple compounds and ingredients. In other embodiments,rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, orrebaudioside G is included and/or added to the consumable products andbeverage products. In certain preferred embodiments, the rebaudioside V,rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G isincluded and/or added at a final concentration that ranges from about 5ppm to about 100 ppm, from about 5 ppm to about 95 ppm, from about 5 ppmto about 90 ppm, from about 5 ppm to about 85 ppm, from about 5 ppm toabout 80 ppm, from about 5 ppm to about 75 ppm, from about 5 ppm toabout 70 ppm, from about 5 ppm to about 65 ppm, from about 5 ppm toabout 60 ppm, from about 5 ppm to about 55 ppm, from about 5 ppm toabout 50 ppm, from about 5 ppm to about 45 ppm, from about 5 ppm toabout 40 ppm, from about 5 ppm to about 35 ppm, from about 5 ppm toabout 30 ppm, from about 5 ppm to about 25 ppm, from about 5 ppm toabout 20 ppm, from about 5 ppm to about 15 ppm, or from about 5 ppm toabout 10 ppm. Alternatively, the rebaudioside V or rebaudioside W isincluded and/or added at a final concentration that ranges from about 5ppm to about 100 ppm, from about 10 ppm to about 100 ppm, from about 15ppm to about 100 ppm, from about 20 ppm to about 100 ppm, from about 25ppm to about 100 ppm, from about 30 ppm to about 100 ppm, from about 35ppm to about 100 ppm, from about 40 ppm to about 100 ppm, from about 45ppm to about 100 ppm, from about 50 ppm to about 100 ppm, from about 55ppm to about 100 ppm, from about 60 ppm to about 100 ppm, from about 65ppm to about 100 ppm, from about 70 ppm to about 100 ppm, from about 75ppm to about 100 ppm, from about 80 ppm to about 100 ppm, from about 85ppm to about 100 ppm, from about 90 ppm to about 100 ppm, or from about95 ppm to about 100 ppm. For example, rebaudioside V or rebaudioside Wmay be included and/or added at a final concentration of about 5 ppm,about 10 ppm, about 15 ppm, about 20 ppm, about 25 ppm, about 30 ppm,about 35 ppm, about 40 ppm, about 45 ppm, about 50 ppm, about 55 ppm,about 60 ppm, about 65 ppm, about 70 ppm, about 75 ppm, about 80 ppm,about 85 ppm, about 90 ppm, about 95 ppm, or about 100 ppm, includingany range in between these values.

In certain embodiments, rebaudioside V, rebaudioside W, rebaudioside KA,rebaudioside M, or rebaudioside G is the only sweetener included and/oradded to the consumable products and the beverage products. In suchembodiments, the consumable products and the beverage products have asweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrosesolution, about 1% to about 3% (w/v-%) sucrose solution, or about 1% toabout 2% (w/v-%) sucrose solution. Alternatively, the consumableproducts and the beverage products have a sweetness intensity equivalentto about 1% to about 4% (w/v-%) sucrose solution, about 2% to about 4%(w/v-%) sucrose solution, about 3% to about 4% (w/v-%) sucrose solution,or about 4%. For example, the consumable products and the beverageproducts may have a sweetness intensity equivalent to about 1%, about2%, about 3%, or about 4% (w/v-%) sucrose solution, including any rangein between these values.

The consumable products and beverage products of the present disclosurecan include a mixture of rebaudioside V, rebaudioside W, rebaudiosideKA, rebaudioside M, or rebaudioside G and one or more sweeteners of thepresent disclosure in a ratio sufficient to achieve a desirablesweetness intensity, nutritional characteristic, taste profile,mouthfeel, or other organoleptic factor.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1

In this Example, full-length DNA fragments of all candidate UGT geneswere synthesized.

Specifically, the cDNAs were codon optimized for E. coli expression(Genscript, Piscataway, N.J.). The synthesized DNA was cloned into abacterial expression vector pETite N-His SUMO Kan Vector (Lucigen). Forthe nucleotide sequence encoding the UDP-glycosyltransferase fusionenzymes (UGT76G1-AtSUS1 and EUGT11-AtSUS1), a GSG-linker (encoded by thenucleotide sequence: ggttctggt) was inserted in frame between anucleotide sequence encoding the uridine diphospho glycosyltransferasedomain and the nucleotide sequence encoding the sucrose synthase 1 fromA. thaliana (AtSUS1). Table 2 summarizes the protein and sequenceidentifier numbers.

TABLE 2 Sequence Identification Numbers. Name SEQ ID NO DescriptionUGT76G1 SEQ ID NO: 1 Amino acid UGT76G1 SEQ ID NO: 2 Nucleic acid EUGT11SEQ ID NO: 3 Amino acid EUGT11 SEQ ID NO: 4 Nucleic acid HV1 SEQ ID NO:5 Amino acid HV1 SEQ ID NO: 6 Nucleic acid AtSUS1 SEQ ID NO: 7 Aminoacid AtSUS1 SEQ ID NO: 8 Nucleic acid GS fusion enzyme SEQ ID NO: 9Amino acid GS fusion enzyme SEQ ID NO: 10 Nucleic acid EUS fusion enzymeSEQ ID NO: 11 Amino acid EUS fusion enzyme SEQ ID NO: 12 Nucleic acid

Each expression construct was transformed into E. coli BL21 (DE3), whichwas subsequently grown in LB media containing 50 μg/mL kanamycin at 37°C. until reaching an OD₆₀₀ of 0.8-1.0. Protein expression was induced byaddition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and theculture was further grown at 16° C. for 22 hr. Cells were harvested bycentrifugation (3,000×g; 10 min; 4° C.). The cell pellets were collectedand were either used immediately or stored at −80° C.

The cell pellets were re-suspended in lysis buffer (50 mM potassiumphosphate buffer, pH 7.2, 25 μg/ml lysozyme, 5 μg/ml DNase I, 20 mMimidazole, 500 mM NaCl, 10% glycerol, and 0.4% TRITON X-100). The cellswere disrupted by sonication at 4° C., and the cell debris was clarifiedby centrifugation (18,000×g; 30 min). Supernatant was loaded to aequilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen)affinity column. After loading of protein sample, the column was washedwith equilibration buffer to remove unbound contaminant proteins. TheHis-tagged UGT recombinant polypeptides were eluted by equilibrationbuffer containing 250 mM imidazole. Purified HV1 (61.4 kD), UGT76G1(65.4 kD), AtSUS1 (106.3 kD), EUGT11 (62 kD), UGT76G1-SUS1 (GS) (157.25kD) and EUGT11-AtSUS1 (155 kD) fusion proteins were shown in FIG. 2.

Example 2

In this Example, candidate UGT recombinant polypeptides were assayed forglycosyltranferase activity by using tested steviol glycosides as thesubstrate.

Typically, the recombinant polypeptide (10 μg) was tested in a 200 μl invitro reaction system. The reaction system contains 50 mM potassiumphosphate buffer, pH 7.2, 3 mM MgCl₂, 1 mg/ml steviol glycosidesubstrate, 1 mM UDP-glucose. The reaction was performed at 30° C. andterminated by adding 200 μL 1-butanol. The samples were extracted threetimes with 200 μL 1-butanol. The pooled fraction was dried and dissolvedin 70 μL 80% methanol for high-performance liquid chromatography (HPLC)analysis. Rubusoside (99%, Blue California, Calif.), purified Reb G(98.8%), Reb KA (98.4%) and Reb V (80%) was used as substrate in invitro reactions.

The UGT catalyzed glycosylation reaction was be coupled to a UDP-glucosegenerating reaction catalyzed by a sucrose synthase (such as AtSUS1). Inthis method, the UDP-glucose was generated from sucrose and UDP, suchthat the addition of extra UDP-glucose can be omitted. In the assay,recombinant AtSUS1 was added in UGT reaction system and UDP-glucose canbe regenerated from UDP. AtSUS1 sequence (Bieniawska et al., Plant J.2007, 49: 810-828) was synthesized and inserted into a bacterialexpression vector. The recombinant AtSUS1 protein was expressed andpurified by affinity chromatography.

HPLC analysis was performed using a Dionex UPLC ultimate 3000 system(Sunnyvale, Calif.), including a quaternary pump, a temperaturecontrolled column compartment, an auto sampler and a UV absorbancedetector. Phenomenex Luna NH₂, Luna C18 or Synergi Hydro-RP column withguard column was used for the characterization of steviol glycosides.Acetonitrile in water or in Na₃PO₄ buffer was used for isocratic elutionin HPLC analysis. The detection wavelength was 210 nm.

Example 3

In this Example, the recombinant HV1 polypeptides were analyzed fortransferring a sugar moiety to rubusoside to produce rebaudioside KA(“Minor diterpene glycosides from the leaves of Stevia rebaudiana”.Journal of Natural Products (2014), 77(5), 1231-1235) in all reactionconditions with or without AtSUS1.

As shown in FIG. 3, the recombinant HV1 polypeptides transferred a sugarmoiety to rubusoside to produce Reb KA in all reaction conditions withor without AtSUS1. Rubusoside was completely converted to Reb KA and RebE by the recombinant HV1 in a UGT-SUS coupling reaction system (G, I).However, only partial rubososide was converted to Reb KA after 24 hours(H) by the recombinant HV1 polypeptide alone without being coupled toAtSUS1, indicating AtSUS1 enhanced the conversion efficiency in theUGT-SUS coupling system. In the HV1-AtSUS1 coupling reaction system,produced Reb KA can be continually converted to Reb E.

Example 4

In this Example, HV1 activity was analyzed using Reb E as a substrate.

Reb E substrate (0.5 mg/ml) was incubated with the recombinant HV1polypeptide (20 μg) and AtSUS1 (20 μg) in a UGT-SUS coupling reactionsystem (200 μL) under conditions similar to those used in the examplesabove. As shown in FIG. 4, Reb Z was produced by the combination of therecombinant HV1 polypeptide and AtSUS1. These results indicated that HV1can transfer a glucose moiety to Reb E to form RZ. FIG. 4 showsrebaudioside Z (“Reb Z”) can be produced from rebaudioside E (“Reb E”)catalyzed by a recombinant HV1 polypeptide and a recombinant AtSUS1 in aHV1-AtSUS1 coupling reaction system. HV1 can transfer a glucose to Reb Eto produce Reb Z, mixture of Reb Z1 and Reb Z2 in the ratio between60:40 to 70:30 (U.S. Provisional Application No. 61/898,571, assigned toConagen Inc.).

Example 5

In this Example, to confirm the conversion of Reb KA to Reb E, purifiedReb KA substrate was incubated with recombinant HV1 with or withoutAtSUS1. As shown in FIG. 5, Reb E was produced by the recombinant HV1polypeptide in both reaction conditions. However, AtSUS1 polypeptide ina UGT-SUS coupling reaction system can enhance the reaction efficiency.All Reb KA substrate can be completely converted to Reb E in the UGT-SUScoupling system (D).

Example 6

In this Example, EUGT11 activity was analyzed using rubusoside as asubstrate.

As shown in FIG. 6, EUGT11 can transfer a sugar moiety to rubusoside toproduce Reb KA and stevioside in all reaction conditions with or withoutAtSUS1. AtSUS1 enhanced the conversion efficiency in the UGT-SUScoupling system. In the HV1-AtSUS1 coupling reaction system, Reb E canbe continually converted by EUGT11. EUS fusion protein exhibited higheractivity under same reaction condition. All produced Reb KA andstevioside was completely converted to Reb E by EUS at 48 hr. Reb E canbe continually converted to Reb D3.

Example 7

In this Example, EUGT11 activity was analyzed using Reb KA as asubstrate.

EUGT11 is a UGT with a 1,2-19-O-glucose glycosylation activity toproduce related steviol glycoside (PCT Published ApplicationWO2013/022989, assigned to Evolva SA). For example, EUGT11 can catalyzethe reaction to produce Reb E from stevioside. EUGT11 also has a1,6-13-O-glucose glycosylation activity that can transfer a glucosemolecule to rebaudioside E to form rebaudioside D3 (U.S. patentapplication Ser. No. 14/269,435, assigned to Conagen, Inc.). In theexperiments, we found EUGT11 can transfer a glucose residue to Reb KA toform Reb E. As shown in FIG. 7, EUGT11 can transfer a sugar moiety toReb KA to produce Reb E in all reaction conditions with (E, H) orwithout AtSUS1 (D, G). AtSUS1 enhanced the conversion efficiency in theUGT-SUS coupling system (E, H). In the EUGT11-AtSUS1 coupling reactionsystem (E, H) and EUS fusion reaction system (F, I), all Reb KA wascompletely converted and the produced Reb E can be continually convertedto Reb D3.

Example 8

In this Example, UGT76G1 activity was analyzed using rubusoside as asubstrate.

UGT76G1 has 1,3-13-O-glucose glycosylation activity that can transfer aglucose molecule to stevioside to form rebaudioside A and to Reb E toform rebaudioside D. In the example, we found UGT76G1 can transfer aglucose residue to rubusoside to form rebaudioside G.

As shown in FIG. 8, UGT76G1 can transfer a sugar moiety to rubusoside toproduce Reb G in all reaction conditions with (D, G) or without AtSUS1(C, F). AtSUS1 enhanced the conversion efficiency in the UGT-SUScoupling system. GS fusion protein exhibited higher activity under samereaction condition (E, H). All rubusoside was completely converted toReb G by GS at 12 hr (E).

Example 9

In this Example, UGT76G1 activity was analyzed using rebaudioside KA asa substrate.

To further identify the enzymatic activity of UGT76G1, an in vitro assaywas performed using rebaudioside KA as substrate. Surprisingly, a novelsteviol glycoside (rebaudioside V “Reb V”) was produced in an early timepoint. At later time points, Reb V produced in the reaction wasconverted to another novel steviol glycoside (rebaudioside W “RebW”).

As shown in FIG. 9, UGT76G1 can transfer a sugar moiety to Reb KA toproduce Reb V in all reaction conditions with (F, I) or without AtSUS1(E, H). AtSUS1 enhanced the conversion efficiency in the UGT-SUScoupling system (F, I). In the UGT76G1-AtSUS1 coupling reaction system(I) and GS fusion reaction system (J), produced Reb V was completelyconverted to Reb W at 12 hr.

Example 10

In this Example, UGT76G1 activity was analyzed using Reb V as asubstrate.

Purified Reb V as substrate was introduced into the reaction system. Asshown in FIG. 10C, Reb V was surprisingly completely converted to Reb Wby the UGT76G1 recombinant polypeptide in UGT-SUS1 coupling system at 6hr.

Example 11

In this Example, HV1 activity was analyzed using Reb G as a substrate.

As shown in FIG. 11, the recombinant HV1 polypeptides transferred asugar moiety to rebaudioside G to produce Reb V in all reactionconditions with or without AtSUS1. Reb G was completely converted to RebV by the recombinant HV1 in a UGT-SUS coupling reaction system (E, G).However, only partial Reb G was converted to Reb V after 24 hours (F) bythe recombinant HV1 polypeptide alone without being coupled to AtSUS1,indicating AtSUS1 enhanced the conversion efficiency in the UGT-SUScoupling system.

Example 12

In this Example, EUGT11 activity was analyzed using Reb G as asubstrate.

As shown in FIG. 12, the recombinant EUGT11 polypeptides transferred asugar moiety to rebaudioside G to produce Reb V in all reactionconditions with (F, I) or without AtSUS1 (E, H). More Reb G wasconverted to Reb V by the recombinant EUGT11 in a UGT-SUS couplingreaction system (F, I). However, only partial Reb G was converted to RebV by the recombinant EUGT11 polypeptide alone without being coupled toAtSUS1 (E, H), indicating AtSUS1 enhanced the conversion efficiency inthe UGT-SUS coupling system. EUS fusion protein exhibited higheractivity under same reaction condition (G, J). All Reb G in the reactionsystem was completely converted to Reb V by EUS at 24 hr (J).

Example 13

In this Example, HV1 combined with UGT76G1 activities were analyzedusing rubusoside as a substrate.

Rebusoside substrate was incubated with the recombinant HV1 polypeptide,UGT76G1, and AtSUS1 in a UGT-SUS coupling reaction system underconditions similar to those used in the examples above. The productswere analyzed by HPLC. As shown in FIG. 13, Reb V and Reb W was producedby the combination of the recombinant HV1 polypeptide, UGT76G1, andAtSUS1. Thus, the recombinant HV1 polypeptide, which showed a1,2-19-O-glucose and 1,2-13-O-glucose glycosylation activity, can beused in combination with other UGT enzymes (such as UGT76G1, whichshowed a 1,3-13-O-glucose and 1,3-19-O-glucose glycosylation activity)for the complex, multi-step biosynthesis of steviol glycosides. If HV1recombinant protein was combined with GS fusion protein in the reactionsystem, Reb V and Reb W was also produced by these UGT enzymes,indicating UGT-SUS coupling reaction can be generated by the GS fusionprotein.

Example 14

In this Example, EUGT11 combined with UGT76G1 activities were analyzedusing rubusoside as a substrate.

Rebusoside substrate was incubated with the recombinant EUGT11polypeptide, UGT76G1, and AtSUS1 in a UGT-SUS coupling reaction systemunder conditions similar to those used in the examples above. Theproducts were analyzed by HPLC. As shown in FIG. 14, Reb W was producedby the combination of the recombinant EUGT11 polypeptide, UGT76G1, andAtSUS1. Thus, the recombinant EUGT11 polypeptide, which showed a 1,2-19-O-glucose and 1, 2-13-O-glucose glycosylation activity, can be usedin combination with other UGT enzymes (such as UGT76G1, which showed a1,3-13-O-glucose and 1,3-19-O-Glucose glycosylation activity) for thecomplex, multi-step biosynthesis of steviol glycosides. If EUGT11recombinant protein was combined with GS fusion protein in the reactionsystem, Reb W was also produced by these UGT enzymes, indicating UGT-SUScoupling reaction can be generated by the GS fusion protein.

Example 15

In this Example, HV1 combined with UGT76G1 activities were analyzedusing Reb G as a substrate.

Reb G substrate was incubated with the recombinant HV1 polypeptide,UGT76G1, and AtSUS1 in a UGT-SUS coupling reaction system underconditions similar to those used in the examples above. The productswere analyzed by HPLC. As shown in FIG. 15, Reb V and Reb W was producedby the combination of the recombinant HV1 polypeptide, UGT76G1, andAtSUS1. After 12 hours, all rubusoside substrate was converted to Reb V,and after 36 hours, all produced Reb V was converted to Reb W. Thus, therecombinant HV1 polypeptide, which showed a 1,2-19-O-glucose and1,2-13-O-glucose glycosylation activity, can be used in combination withother UGT enzymes (such as UGT76G1, which showed a 1,3-13-O-glucose and1,3-19-O-Glucose glycosylation activity) for the complex, multi-stepbiosynthesis of steviol glycosides. If HV1 recombinant protein wascombined with GS fusion protein in the reaction system, Reb V and Reb Wwas also produced by these UGT enzymes, indicating UGT-SUS couplingreaction can be generated by the GS fusion protein.

Example 16

In this Example, EUGT11 combined with UGT76G1 activities were analyzedusing Reb G as a substrate.

Reb G substrate was incubated with the recombinant EUGT11 polypeptide,UGT76G1, and AtSUS1 in a UGT-SUS coupling reaction system underconditions similar to those used in the examples above. The productswere analyzed by HPLC. As shown in FIG. 16, Reb W was produced by thecombination of the recombinant EUGT11 polypeptide, UGT76G1, and AtSUS1.Thus, the recombinant EUGT11 polypeptide, which showed a 1,2-19-O-glucose and 1, 2-13-O-glucose glycosylation activity, can be usedin combination with other UGT enzymes (such as UGT76G1, which showed a1,3-13-O-glucose and 1,3-19-O-Glucose glycosylation activity) for thecomplex, multi-step biosynthesis of steviol glycosides. If EUGT11recombinant protein was combined with GS fusion protein in the reactionsystem, Reb W was also produced by these UGT enzymes, indicating UGT-SUScoupling reaction can be generated by the GS fusion protein.

Example 17

In this Example, UGT76G1 and GS fusion enzyme activity was analyzedusing Reb D as a substrate.

Reb D substrate was incubated with the recombinant UGT76G1 underconditions similar to those used in the examples above. The productswere analyzed by HPLC. As shown in FIG. 22, Reb M was produced by theUGT76G1 with (FIGS. 22 D and G) or without AtSUS1 (FIGS. 22 C and F) inthe reactions. Thus, the recombinant UGT76G1 polypeptide, which showed a1, 3-19-O-glucose glycosylation activity, can be used in biosynthesis ofrebaudioside M. Reb D was completely converted to Reb M by therecombinant UGT76G1 in a UGT-SUS coupling reaction system (FIG. 22 G).However, only partial Reb D was converted to Reb M after 6 hours (F) bythe recombinant UGT76G1 polypeptide alone without being coupled toAtSUS1, indicating AtSUS1 enhanced the conversion efficiency in theUGT-SUS coupling system. GS fusion protein exhibited similar activity asUGT76G1-AtSUS1 coupling reaction under same reaction condition (E, H).All Reb D was completely converted to Reb M by GS at 6 hr (H),indicating UGT-SUS coupling reaction can be generated by the GS fusionprotein.

Example 18

In this Example, UGT76G1 and GS fusion enzyme activity was analyzedusing Reb E as substrate.

Reb E substrate was incubated with the recombinant UGT76G1 or GS fusionenzyme under conditions similar to those used in the examples above. Theproducts were analyzed by HPLC. As shown in FIG. 23, Reb D was producedby the UGT76G1 with (FIGS. 23 E, H and K) or without AtSUS1 (FIGS. 22 D,G and J) and GS fusion enzyme (FIGS. 23 F, I and L) in the reactions.Furthermore, Reb M was formed from Reb D produced in the reactions.Thus, the recombinant UGT76G1 polypeptide, which showed a1,3-13-O-glucose and 1,3-19-O-glucose glycosylation activity, can beused in the biosynthesis of rebaudioside D and rebaudioside M. Reb E wascompletely converted to Reb M by the recombinant UGT76G1 in a UGT-SUScoupling reaction system after 24 hr (FIG. 23K). However, only Reb D wasconverted from Reb E completely after 24 hours (J) by the recombinantUGT76G1 polypeptide alone without being coupled to AtSUS1, indicatingAtSUS1 enhanced the conversion efficiency in the UGT-SUS coupling systemthrough continuing UDPG production. GS fusion protein exhibited similaractivity as UGT76G1-AtSUS1 coupling reaction under same reactioncondition (FIGS. 23 F, I and L), indicating UGT-SUS coupling reactioncan be generated by the GS fusion protein.

Example 19

In this Example, HV1 combined with UGT76G1 activities were analyzedusing stevioside as a substrate.

Stevioside substrate was incubated with the recombinant HV1 polypeptideand UGT76G1 or GS fusion enzyme under conditions similar to those usedin the examples above. The products were analyzed by HPLC. As shown inFIG. 24, Reb A was produced by the combination of the recombinant HV1polypeptide and UGT76G1 in all reactions. Furthermore, Reb D and Reb Mwere detected in the reactions using the combination of recombinant HV1polypeptide, UGT76G1 polypeptide and AtSUS1 (FIGS. 24 E, H and K) or thecombination of recombinant GS fusion enzyme and HV1 polypeptide (FIGS.24 F, I and L). The recombinant HV1 polypeptide, which showed a 1,2-19-O-glucose glycosylation activity, can be used in combination withother UGT enzymes (such as UGT76G1, which showed a 1,3-13-O-glucose and1,3-19-O-glucose glycosylation activity) for the complex, multi-stepbiosynthesis of rebaudioside D and rebaudioside M. The results alsoshowed that AtSUS1 enhanced the conversion efficiency in the UGT-SUScoupling system through continuing UDPG production (FIGS. 24 E, H andK). GS fusion protein exhibited similar activity as UGT76G1-AtSUS1coupling reaction under same reaction condition (FIGS. 24 F, I and L),indicating UGT-SUS coupling reaction can be generated by the GS fusionprotein.

Example 20

In this Example, HV1 combined with UGT76G1 activities were analyzedusing Reb A as a substrate.

Reb A substrate was incubated with the recombinant HV1 polypeptide andUGT76G1 or GS fusion enzyme under conditions similar to those used inthe examples above. The products were analyzed by HPLC. As shown in FIG.25, Reb D was produced by the combination of the recombinant HV1polypeptide and UGT76G1 in all reactions. Furthermore, Reb M wasdetected in the reactions using the combination of recombinant HV1polypeptide, UGT76G1 polypeptide and AtSUS1 (FIGS. 25 D, G and J) or thecombination of recombinant GS fusion enzyme and HV1 polypeptide (FIGS.25 E, H and K). The recombinant HV1 polypeptide, which showed a 1,2-19-O-glucose glycosylation activity, can be used in combination withother UGT enzymes (such as UGT76G1, which showed a 1,3-19-O-glucoseglycosylation activity) for the complex, multi-step biosynthesis ofrebaudioside D and rebaudioside M. The results also showed that AtSUS1enhanced the conversion efficiency in the UGT-SUS coupling systemthrough continuing UDPG production (FIGS. 25 D, G and J). GS fusionprotein exhibited similar activity as UGT76G1-AtSUS1 coupling reactionunder same reaction condition (FIGS. 25 E, H and K), indicating UGT-SUScoupling reaction can be generated by the GS fusion protein.

Example 21

In this Example, the structure of Reb V was analyzed by NMR.

The material used for the characterization of Reb V was produced byusing enzymatic conversion of Reb G and purified by HPLC. HRMS data weregenerated with a LTQ Orbitrap Discovery HRMS instrument, with itsresolution set to 30 k. Scanned data from m/z 150 to 1500 in positiveion electrospray mode. The needle voltage was set to 4 kV; the othersource conditions were sheath gas=25, aux gas=0, sweep gas=5 (all gasflows in arbitrary units), capillary voltage=30V, capillarytemperature=300° C., and tube lens voltage=75. Sample was diluted with2:2:1 acetonitrile:methanol:water (same as infusion eluent) and injected50 microliters. NMR spectra were acquired on Bruker Avance DRX 500 MHzor Varian INOVA 600 MHz instruments using standard pulse sequences. The1D (¹H and ¹³C) and 2D (TOCSY, HMQC, and HMBC) NMR spectra wereperformed in C₅D₅N.

The molecular formula of Reb V has been deduced as C₄₄H₇₀O₂₃ on thebasis of its positive high resolution (HR) mass spectrum which showedadduct ions corresponding to [M+Na]⁺ at m/z 989.4198; this compositionwas supported by the ¹³C NMR spectral data. The ¹H NMR spectral data ofReb V showed the presence of two methyl singlets at δ 0.97 and 1.40, twoolefinic protons as singlets at δ 5.06 and 5.71 of an exocyclic doublebond, nine sp3 methylene and two sp3 methine protons between δ0.74-2.72, characteristic for the ent-kaurane diterpenoids isolatedearlier from the genus Stevia. The basic skeleton of ent-kauranediterpenoids was supported by the COSY and TOCSY studies which showedkey correlations: H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11;H-11/H-12. The ¹H NMR spectrum of Reb V also showed the presence of fouranomeric protons resonating at δ 5.08, 5.38, 5.57, and 6.23; suggestingfour sugar units in its structure. Acid hydrolysis of Reb V with 5%H₂SO₄ afforded D-glucose which was identified by direct comparison withauthentic sample by TLC. Enzymatic hydrolysis of Reb V furnished anaglycone which was identified as steviol by comparison of ¹H NMR andco-TLC with standard compound. The large coupling constants observed forthe four anomeric protons of the glucose moieties at δ 5.08 (d, J=7.8Hz), 5.38 (d, J=8.1 Hz), 5.57 (d, J=8.0 Hz), and 6.23 (d, J=7.8 Hz),suggested their β-orientation as reported for steviol glycosides. The ¹Hand ¹³C NMR values for Reb V were assigned on the basis of TOCSY, HMQCand HMBC data and are given in Table 3.

TABLE 3 ¹H and ¹³C NMR spectral data (chemical shifts and couplingconstants) for Reb V and Reb G ^(a-c). Reb V Reb G Position ¹H NMR ¹³CNMR ¹H NMR ¹³C NMR  1 0.74 m, 1.66 m 41.1 0.78 m, 1.69 m 41.3  2 1.43 m,2.18 m 20.4 1.44 m, 2.20 m 20.0  3 1.06 m, 2.72 d 38.4 1.05 m, 2.70 d38.8 (12.8) (11.6)  4 — 44.8 — 44.9  5 1.32 m 57.9 1.32 m 57.8  6 1.84m, 2.20 m 22.7 1.87 m, 2.24 m 22.6  7 1.06 m, 1.70 m 42.2 1.07 m, 1.72 m42.2  8 — 42.5 — 43.1  9 0.91 d (7.8) 54.5 0.92 d (7.6) 54.4 10 — 40.2 —40.4 11 1.72 m 21.0 1.75 m 21.2 12 2.18 m, 2.38 m 38.3 2.26 m, 2.38 m37.7 13 — 87.6 — 86.4 14 1.68 m, 2.43 m 44.8 1.78 m, 2.50 m 44.6 15 1.96m, 2.24 m 48.9 2.06 m, 2.32 m 48.2 16 — 153.7 — 155.0 17 5.06 s, 5.71 s105.7 5.00 s, 5.49 s 104.8 18 1.40 s 29.6 1.32 s 28.8 19 — 176.4 — 177.420 0.97 s 16.7 1.25 s 16.2  1′ 6.23 d (7.8) 94.2 6.16 d (7.6) 96.4  2′3.98 m 74.5 4.01 m 74.5  3′ 4.14 m 79.3 4.09 m 79.3  4′ 4.36 m 71.6 4.34m 71.6  5′ 4.24 m 79.9 4.22 m 79.9  6′ 4.06 m, 4.48 m 62.6 4.04 m, 4.4462.6 dd (3.2, 7.6)  1″ 5.08 d (7.8) 99.6 5.06 d (7.4) 99.9  2″ 3.94 m74.7 3.92 m 74.5  3″ 4.04 m 89.3 4.06 m 89.5  4″ 4.28 m 71.2 4.23 m 71.0 5″ 4.00 m 78.2 4.02 m 78.1  6″ 4.24 m, 4.58 m 63.0 4.27 m, 4.56 63.1 dd(2.8, 8.4)  1″′ 5.38 d (8.1) 106.4 5.27 d (8.4) 106.5  2″′ 4.16 m 76.14.14 m 76.0  3″′ 4.34 m 79.2 4.37 m 79.3  4″′ 4.26 m 72.2 4.28 m 72.2 5″′ 3.78 m 78.8 3.89 m 78.8  6″′ 4.14 m, 4.44 m 63.2 4.18 m, 4.48 m63.2  1″″ 5.57 d (8.0) 105.7  2″″ 3.96 m 76.5  3″″ 4.32 m 79.6  4″″ 4.20m 72.5  5″″ 3.87 m 79.0  6″″ 4.12 m, 4.46 m 63.5 ^(a) assignments madeon the basis of TOCSY, HMQC and HMBC correlations; ^(b) Chemical shiftvalues are in δ (ppm); ^(c) Coupling constants are in Hz.

Based on the results from NMR spectral data and hydrolysis experimentsof Reb V, it was concluded that there are four β-D-glucosyl units in itsstructure connected to the aglycone steviol. A close comparison of the¹H and ¹³C NMR values of Reb V with Reb G suggested the presence of asteviol aglycone moiety with a 3-O-β-D-glucobiosyl unit at C-13 in theform of ether linkage and another β-D-glucosyl unit at C-19 position inthe form of an ester linkage, leaving the assignment of the fourthβ-D-glucosyl moiety (FIG. 17). The downfield shift for both the ¹H and¹³C chemical shifts at 2-position of sugar I of the β-D-glucosyl moietysupported the presence of β-D-glucosyl unit at this position. Thestructure was further supported by the key TOCSY and HMBC correlationsas shown in FIG. 18. Based on the results of NMR and mass spectral dataas well as hydrolysis studies, the structure of Reb V produced by theenzymatic conversion of Reb G was deduced as13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

Acid hydrolysis of Reb V. To a solution of Reb V (5 mg) in MeOH (10 ml)was added 3 ml of 5% H₂SO₄ and the mixture was refluxed for 24 hours.The reaction mixture was then neutralized with saturated sodiumcarbonate and extracted with ethyl acetate (EtOAc) (2×25 ml) to give anaqueous fraction containing sugars and an EtOAc fraction containing theaglycone part. The aqueous phase was concentrated and compared withstandard sugars using the TLC systems EtOAc/n-butanol/water (2:7:1) andCH₂Cl₂/MeOH/water (10:6:1); the sugars were identified as D-glucose.

Enzymatic hydrolysis of Reb V. Reb V (1 mg) was dissolved in 10 ml of0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillusniger (50 uL, Sigma-Aldrich, P2736) was added. The mixture was stirredat 50° C. for 96 hr. The product precipitated out during the reactionfrom the hydrolysis of 1 was identified as steviol by comparison of itsco-TLC with standard compound and ¹H NMR spectral data. A compound namedReb V was confirmed as13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester on the basis of extensive 1D and 2D NMR as well as high resolutionmass spectral data and hydrolysis studies.

Example 22

In this Example, the structure of Reb W was analyzed by NMR.

The material used for the characterization of Reb W was produced byusing enzymatic conversion of Reb V and purified by HPLC. HRMS data weregenerated with a LTQ Orbitrap Discovery HRMS instrument, with itsresolution set to 30 k. Scanned data from m/z 150 to 1500 in positiveion electrospray mode. The needle voltage was set to 4 kV; the othersource conditions were sheath gas=25, aux gas=0, sweep gas=5 (all gasflows in arbitrary units), capillary voltage=30V, capillarytemperature=300 C, and tube lens voltage=75. Sample was diluted with2:2:1 acetonitrile:methanol:water (same as infusion eluent) and injected50 microliters. NMR spectra were acquired on Bruker Avance DRX 500 MHzor Varian INOVA 600 MHz instruments using standard pulse sequences. The1D (¹H and ¹³C) and 2D (TOCSY, HMQC, and HMBC) NMR spectra wereperformed in C₅D₅N.

The molecular formula of Reb W has been deduced as C₅₀H₈₀O₂₈ on thebasis of its positive high resolution (HR) mass spectrum which showedadduct ions corresponding to [M+Na]⁺ at m/z 1151.4708; this compositionwas supported by the ¹³C NMR spectral data. The ¹H NMR spectral data ofReb W showed the presence of two methyl singlets at δ 0.92 and 1.39, twoolefinic protons as singlets at δ 5.10 and 5.73 of an exocyclic doublebond, nine sp3 methylene and two sp3 methine protons between δ0.72-2.72, characteristic for the ent-kaurane diterpenoids isolatedearlier from the genus Stevia. The basic skeleton of ent-kauranediterpenoids was supported by the TOCSY studies which showed keycorrelations: H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12.The ¹H NMR spectrum of Reb W also showed the presence of five anomericprotons resonating at δ 5.10, 5.34, 5.41, 5.81, and 6.14; suggestingfive sugar units in its structure. Acid hydrolysis of Reb W with 5%H₂SO₄ afforded D-glucose which was identified by direct comparison withauthentic sample by TLC. Enzymatic hydrolysis of Reb W furnished anaglycone which was identified as steviol by comparison of ¹H NMR andco-TLC with standard compound. The large coupling constants observed forthe five anomeric protons of the glucose moieties at δ 5.10 (d, J=7.4Hz), 5.34 (d, J=7.9 Hz), 5.41 (d, J=7.9 Hz), 5.89 (d, J=7.9 Hz), and6.14 (d, J=7.9 Hz), suggested their β-orientation as reported forsteviol glycosides [1-5, 9-13]. The ¹H and ¹³C NMR values for Reb W wereassigned on the basis of TOCSY, HMQC and HMBC data and are given inTable 4.

TABLE 4 ¹H and ¹³C NMR spectral data (chemical shifts and couplingconstants) for Reb W and Reb V ^(a-c). Reb W Reb V Position ¹H NMR ¹³CNMR ¹H NMR ¹³C NMR  1 0.72 m, 1.67 m 41.0 0.78 m, 1.69 m 41.1  2 1.42 m,2.18 m 20.4 1.44 m, 2.20 m 20.4  3 1.06 m, 2.72 d 38.6 1.05 m, 2.70 d38.4 (13.4) (11.6)  4 — 44.8 — 44.8  5 1.34 m 57.9 1.32 m 57.9  6 1.84m, 2.18 m 22.8 1.87 m, 2.24 m 22.7  7 1.07 m, 1.69 m 42.3 1.07 m, 1.72 m42.2  8 — 42.4 — 42.5  9 0.90 d (5.8) 54.5 0.92 d (7.6) 54.5 10 — 40.1 —40.2 11 1.66 m 21.0 1.75 m 21.0 12 2.20 m, 2.39 m 38.3 2.26 m, 2.38 m38.3 13 — 87.8 — 87.6 14 1.63 m, 2.06 m 44.8 1.78 m, 2.50 m 44.8 15 2.06m, 2.04 m 48.8 2.06 m, 2.32 m 48.9 16 — 153.5 — 153.7 17 5.10 s, 5.73 s105.9 5.00 s, 5.49 s 105.7 18 1.39 s 29.4 1.32 s 29.6 19 — 176.5 — 176.420 0.92 s 16.6 1.25 s 16.7  1′ 6.14 d (7.9) 94.1 6.16 d (7.6) 94.2  2′3.98 m 79.6 4.01 m 80.7  3′ 4.20 m 88.9 4.09 m 79.3  4′ 4.34 m 70.0 4.34m 71.2  5′ 4.24 m 79.4 4.22 m 79.9  6′ 4.02 m, 4.39 62.6 4.04 m, 4.4462.6 dd (3.2, 7.6)  1″ 5.10 d (7.4) 99.5 5.06 d (7.4) 99.6  2″ 3.90 m74.7 3.92 m 74.7  3″ 4.04 m 89.3 4.06 m 89.3  4″ 4.25 m 70.4 4.23 m 70.3 5″ 3.98 m 78.6 4.02 m 78.2  6″ 4.27 m, 4.54 m 62.9 4.27 m, 4.56 63.0 dd(2.8, 8.4)  1″′ 5.34 d (7.9) 106.3 5.27 d (8.4) 106.4  2″′ 4.12 m 76.14.14 m 76.1  3″′ 4.33 m 79.2 4.37 m 79.2  4″′ 4.25 m 72.1 4.28 m 72.2 5″′ 3.88 m 78.8 3.89 m 78.8  6″′ 4.16 m, 4.53 m 63.0 4.18 m, 4.48 m63.2  1″″ 5.41 d (7.9) 105.3 5.27 d (8.4) 105.7  2″″ 4.12 m 73.4 4.14 m76.5  3″″ 4.28 m 88.9 4.37 m 79.6  4″″ 4.20 m 72.1 4.28 m 72.5  5″″ 3.78m 79.0 3.89 m 79.0  6″″ 4.08 m, 4.42 m 62.9 4.18 m, 4.48 m 63.5  1″″′5.81 d (7.9) 104.0  2″″′ 4.09 m 77.2  3″″′ 4.24 m 79.3  4″″′ 4.14 m 72.0 5″″′ 3.76 m 79.2  6″″′ 4.04 m, 4.36 m 62.3 ^(a) assignments made on thebasis of TOCSY, HSQC and HMBC correlations; ^(b) Chemical shift valuesare in δ (ppm); ^(c) Coupling constants are in Hz.

Based on the results from NMR spectral data and hydrolysis experimentsof Reb W, it was concluded that there are five β-D-glucosyl units in itsstructure connected to the aglycone steviol. A close comparison of the¹H and ¹³C NMR values of Reb W with Reb V suggested the presence of asteviol aglycone moiety with a 3-O-β-D-glucobiosyl unit at C-13 in theform of ether linkage and a 2-O-β-D-glucobiosyl unit at C-19 position inthe form of an ester linkage, leaving the assignment of the fifthβ-D-glucosyl moiety (FIG. 19). The downfield shift for both the ¹H and¹³C chemical shifts at 3-position of sugar I of the β-D-glucosyl moietysupported the presence of β-D-glucosyl unit at this position. Thestructure was further supported by the key TOCSY and HMBC correlationsas shown in FIG. 20. Based on the results of NMR and mass spectral dataas well as hydrolysis studies, the structure of Reb W produced by theenzymatic conversion of Reb V was deduced as13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

Acid hydrolysis of Reb W. To a solution of Reb W (5 mg) in MeOH (10 ml)was added 3 ml of 5% H₂SO₄ and the mixture was refluxed for 24 hours.The reaction mixture was then neutralized with saturated sodiumcarbonate and extracted with ethyl acetate (EtOAc) (2×25 ml) to give anaqueous fraction containing sugars and an EtOAc fraction containing theaglycone part. The aqueous phase was concentrated and compared withstandard sugars using the TLC systems EtOAc/n-butanol/water (2:7:1) andCH₂Cl₂/MeOH/water (10:6:1); the sugars were identified as D-glucose.

Enzymatic hydrolysis of Reb W. Reb W (1 mg) was dissolved in 10 ml of0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillusniger (50 uL, Sigma-Aldrich, P2736) was added. The mixture was stirredat 50° C. for 96 hr. The product precipitated out during the reactionand was filtered and then crystallized. The resulting product obtainedfrom the hydrolysis of Reb W was identified as steviol by comparison ofits co-TLC with standard compound and ¹H NMR spectral data. A compoundnamed Reb W was confirmed as13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester, on the basis of extensive 1D and 2D NMR as well as highresolution mass spectral data and hydrolysis studies.

After NMR analysis, the structures of Reb V and Reb W were identified asnovel steviol glycosides. The above results further demonstrated thatUGT76G1 has not only a 1,3-13-O-glucose glycosylation activity but also1,3-19-O-glucose glycosylation activity.

Example 23

In this Example, the structure of Reb M was analyzed by NMR.

The material used for the characterization of Reb M was produced fromthe enzymatic conversion of Reb D and purified by HPLC. HRMS data weregenerated with a LTQ Orbitrap Discovery HRMS instrument, with itsresolution set to 30 k. Scanned data from m/z 150 to 1500 in positiveion electrospray mode. The needle voltage was set to 4 kV; the othersource conditions were sheath gas=25, aux gas=0, sweep gas=5 (all gasflows in arbitrary units), capillary voltage=30V, capillarytemperature=300 C, and tube lens voltage=75. Sample was diluted with2:2:1 acetonitrile:methanol:water (same as infusion eluent) and injected50 microliters.

NMR spectra were acquired on Bruker Avance DRX 500 MHz or Varian INOVA600 MHz instruments using standard pulse sequences. The 1D (¹H and ¹³C)and 2D (COSY, HMQC, and HMBC) NMR spectra were performed in C₅D₅N.

The molecular formula of compound Reb M has been deduced as C₅₆H₉₀O₃₃ onthe basis of its positive high resolution (HR) mass spectrum whichshowed an [M+NH₄+CH₃CN]⁺ ion at m/z 1349.5964; this composition wassupported by ¹³C NMR spectral data. The ¹H NMR spectrum of Reb M showedthe presence of two methyl singlets at δ 1.35 and 1.42, two olefinicprotons as singlets at δ 4.92 and 5.65 of an exocyclic double bond, ninemethylene and two methine protons between δ 0.77-2.77 characteristic forthe ent-kaurane diterpenoids isolated earlier from the genus Stevia. Thebasic skeleton of ent-kaurane diterpenoids was supported by COSY(H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12) and HMBC(H-1/C-2, C-10; H-3/C-1, C-2, C-4, C-5, C-18, C-19; H-5/C-4, C-6, C-7,C-9, C-10, C-18, C-19, C-20; H-9/C-8, C-10, C-11, C-12, C-14, C-15;H-14/C-8, C-9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations.The ¹H NMR spectrum of Reb M also showed the presence of anomericprotons resonating at δ 5.33, 5.47, 5.50, 5.52, 5.85, and 6.43;suggesting six sugar units in its structure. Enzymatic hydrolysis of RebM furnished an aglycone which was identified as steviol by comparison ofco-TLC with standard compound. Acid hydrolysis of Reb M with 5% H₂SO₄afforded glucose which was identified by direct comparison withauthentic samples by TLC. The ¹H and ¹³C NMR values for selected protonsand carbons in Reb M were assigned on the basis of TOCSY, HMQC and HMBCcorrelations (Table 5).

Based on the results from NMR spectral data of Reb M, it was concludedthat there are six glucosyl units in its structure (FIG. 26). A closecomparison of the ¹H and ¹³C NMR spectrum of Reb M with rebaudioside Dsuggested that Reb M is also a steviol glycoside which has three glucoseresidues that are attached at the C-13 hydroxyl as a 2,3-branchedglucotriosyl substituent and 2-substituted glucobiosyl moiety in theform of an ester at C-19 leaving the assignment of the additionalglucosyl moiety. The key TOCSY and HMBC correlations shown in FIG. 27suggested the placement of the sixth glucosyl moiety at C-3 position ofSugar I. The large coupling constants observed for the six anomericprotons of the glucose moieties at δ 5.33 (d, J=8.4 Hz), 5.47 (d, J=7.8Hz), 5.50 (d, J=7.4 Hz), 5.52 (d, J=7.4 Hz), 5.85 (d, J=7.4 Hz) and 6.43(d, J=7.8 Hz), suggested their β-orientation as reported for steviolglycosides. Based on the results of NMR and mass spectral studies and incomparison with the spectral values of rebaudioside M reported from theliterature, structure of Reb M produced by enzymatic reaction wasassigned as13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

TABLE 5 ¹H and ¹³C NMR spectral data (chemical shifts and couplingconstants) for Reb M produced by enzymatic reaction ^(a-c). Position ¹HNMR ¹³C NMR  1 0.77 t (12.4), 1.78 m 40.7  2 1.35 m, 2.24 m 20.0  3 1.01m, 2.32 m 38.8  4 — 44.7  5 1.08 d (12.4) 57.8  6 2.23 m, 2.45 q (12.8)23.9  7 1.44 m, 1.83 m 43.0  8 — 41.6  9 0.93 d (7.4) 54.7 10 — 40.1 111.68 m, 1.82 m 20.7 12 1.86 m, 2.28 m 38.8 13 — 88.0 14 2.04 m, 2.77 m43.7 15 1.91 m, 2.03 m 46.8 16 — 153.8 17 4.92 s, 5.65 s 105.2 18 1.35 s28.7 19 — 177.4 20 1.42 s 17.2  1′ 6.43 d (7.8) 95.4  2′ 4.54 m 77.3  3′4.58 m 89.1  4′ 4.22 m 70.5  5′ 4.16 m 78.8  6′ 4.18 m, 4.35 m 62.1  1″5.50 d (7.4) 96.7  2″ 4.19 m 81.9  3″ 5.03 m 88.4  4″ 4.12 m 70.8  5″3.98 m 78.1  6″ 4.22 m, 4.36 m 62.9  1″′ 5.52 d (7.4) 105.4  2″′ 4.24 m76.0  3″′ 4.16 m 78.9  4″′ 4.02 m 73.6  5″′ 3.78 ddd (2.8, 6.4, 9.4)78.0  6″′ 4.32 m, 4.54 m 64.4  1″″ 5.47 d (7.8) 104.4  2″″ 4.00 m 75.9 3″″ 4.40 m 78.2  4″″ 4.12 m 71.6  5″″ 3.96 m 78.4  6″″ 4.20 m, 4.32 m62.5  1″″′ 5.85 d (7.4) 104.7  2″″′ 4.20 m 75.9  3″″′ 4.30 m 78.9  4″″′4.14 m 73.7  5″″′ 3.94 ddd (2.8, 6.4, 9.9) 78.3  6″″′ 4.32 m, 4.67 d(10.6) 64.4  1″″″ 5.33 d (8.4) 104.6  2″″″ 3.98 m 76.2  3″″″ 4.43 m 78.5 4″″″ 4.16 m 71.7  5″″″ 3.88 ddd (2.1, 6.4, 9.4) 78.9  6″″″ 4.10 m, 4.35m 62.5 ^(a) assignments made on the basis of TOCSY, HSQC and HMBCcorrelations; ^(b) Chemical shift values are in δ (ppm); ^(c) Couplingconstants are in Hz.

Acid hydrolysis of compound 1: To a solution of produced Reb M (5 mg) inMeOH (10 ml) was added 3 ml of 5% H₂SO₄ and the mixture was refluxed for24 hours. The reaction mixture was then neutralized with saturatedsodium carbonate and extracted with ethyl acetate (EtOAc) (2×25 ml) togive an aqueous fraction containing sugars and an EtOAc fractioncontaining the aglycone part. The aqueous phase was concentrated andcompared with standard sugars using the TLC systemsEtOAc/n-butanol/water (2:7:1) and CH₂Cl₂/MeOH/water (10:6:1); the sugarswere identified as D-glucose.

Enzymatic hydrolysis of compound: produced Reb M (1 mg) was dissolved in10 ml of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase fromAspergillus niger (50 uL, Sigma-Aldrich, P2736) was added. The mixturewas stirred at 50° C. for 96 hr. The product precipitated out during thereaction from the hydrolysis of 1 was identified as steviol bycomparison of its co-TLC with standard compound and ¹H NMR spectraldata.

A compound named rebaudisode M (Reb M) was obtained was produced bybio-conversion. The complete ¹H and ¹³C NMR spectral assignments forrebaudioside M (Reb M) were made on the basis of extensive 1D and 2D NMRas well as high resolution mass spectral data, which suggested thestructure as13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

Example 24

In this Example, the biosynthesis pathway of steviol glycosides isdiscussed.

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D depict a scheme illustratingthe novel pathways of steviol glycoside biosynthesis from rubusoside. Asdescribed herein, the recombinant HV1 polypeptide (“HV1”) contains a1,2-O-glucose glycosylation activity which transfers a second glucosidemoiety to the C-2′ of 19-O-glucose of rubusoside to produce rebaudiosideKA (“Reb KA”); the recombinant EUGT11 polypeptide (“EUGT11”) contains a1,2-O-glucose glycosylation activity which transfers a second glucosemoiety to the C-2′ of 19-O-glucose of rubusoside to produce rebaudiosideKA; or transfer a second glucose moiety to the C-2′ of 13-O-glucose ofrubusoside to produce stevioside; the recombinant UGT76G1 enzyme(“UGT76G1”) contains a 1,3-O-glucose glycosylation activity whichtransfer a second glucose moiety to the C-3′ of 13-O-glucose ofrubusoside to produce rebaudioside G (“Reb G”). Both of HV1 and EUGT11transfer a second sugar moiety to the C-2′ of 19-O-glucose ofrebaudioside G to produce rebaudioside V (“Reb V”), or transfer a secondglucose moiety to the C-2′ of 13-O-glucose of rebaudioside KA to producerebaudioside E (“Reb E”). FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21Dalso show that a recombinant UGT76G1 enzyme catalyzes the reaction thattransfers the third sugar moiety to C-3′ of the C-19-O-glucose ofrebaudioside V to produce rebaudioside W (“Reb W”) and EUGT11 cancontinually transfer the third glucose moiety to C-6′ of theC-13-O-glucose of rebaudioside E to produce rebaudioside D3. HV1 cantransfer the third glucose moiety to C-2′ of the C-13-O-glucose ofrebaudioside E to produce rebaudioside Z1 (“Reb Z1”), and can transferthe third glucose moiety to C-2′ of the C-19-O-glucose of rebaudioside Eto produce rebaudioside Z2 (“Reb Z2”). Both of HV1 and EUGT11 cancatalyze the conversion of stevioside to Reb E and the conversion ofrebaudioside A (“Reb A”) to rebaudioside D (“Reb D”). UGT76G1 cantransfer the third glucose moiety to C-3′ of the C-13-O-glucose ofrebaudioside E (“Reb E”) to form rebaudioside D (“Reb D”). UGT76G1 alsocatalyze the conversion of stevioside to rebaudioside (“Reb A”) and theconversion of rebaudioside D (“Reb D”) to rebaudioside M (“Reb M”).

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and systems withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

When introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

What is claimed is:
 1. A method for synthesizing rebaudioside E fromrebaudioside KA, the method comprising: (i) preparing a reaction mixturecomprising (a) at least one of rebaudioside KA and stevioside, (b)substrates selected from the group consisting of sucrose, uridinediphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and (c)a UDP-glycosyltransferase fusion enzyme comprising an amino acidsequence having at least 90% identity to SEQ ID NO: 11; (ii) incubatingthe reaction mixture for a sufficient time to produce rebaudioside E,wherein a glucose is covalently coupled to the C2′-13-O-glucose ofrebaudioside KA to produce rebaudioside E.
 2. The method of claim 1,wherein the reaction mixture comprises sucrose and UDP.
 3. The method ofclaim 2, wherein UDP-glucose is generated from sucrose and UDP presentin the reaction mixture by the UDP-glycosyltransferase fusion enzyme. 4.The method of claim 1, wherein the UDP-glycosyltransferase fusion enzymecomprises an amino acid sequence having at least 95% identity to SEQ IDNO:
 11. 5. The method of claim 1, wherein the UDP-glycosyltransferasefusion enzyme is expressed in a host organism.
 6. The method of claim 5,wherein the host organism is a bacterial cell.
 7. The method of claim 5,wherein the host organism is a yeast cell.
 8. The method of claim 5,wherein the host organism is an E. coli cell.
 9. A method forsynthesizing rebaudioside E from rubusoside, the method comprising: (i)preparing a reaction mixture comprising (a) rubusoside, (b) substratesselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose), and (c) aUDP-glycosyltransferase fusion enzyme comprising an amino acid sequencehaving at least 90% identity to SEQ ID NO: 11; (ii) incubating thereaction mixture for a sufficient time to produce rebaudioside E,wherein a glucose is covalently coupled to rubusoside to producerebaudioside KA or stevioside, and a glucose is covalently coupled torebausiode KA or stevioside to produce rebaudioside E.
 10. The method ofclaim 9, wherein the reaction mixture comprises sucrose and UDP.
 11. Themethod of claim 10, wherein UDP-glucose is generated from sucrose andUDP present in the reaction mixture by the UDP-glycosyltransferasefusion enzyme.
 12. The method of claim 9, wherein theUDP-glycosyltransferase fusion enzyme comprises an amino acid sequencehaving at least 95% identity to SEQ ID NO:
 11. 13. The method of claim9, wherein the UDP-glycosyltransferase fusion enzyme is expressed in ahost organism.
 14. The method of claim 13, wherein the host organism isa bacterial cell.
 15. The method of claim 13, wherein the host organismis a yeast cell.
 16. The method of claim 13, wherein the host organismis an E. coli cell.
 17. The method of claim 1, wherein theUDP-glycosyltransferase fusion enzyme comprises the amino acid sequenceof SEQ ID NO:
 11. 18. The method of claim 9, wherein theUDP-glycosyltransferase fusion enzyme comprises the amino acid sequenceof SEQ ID NO: 11.